Background
Pancreatic ductal adenocarcinoma (PDAC) remains one of most aggressive and lethal of human cancers. The overall 5-year survival rate is <5 %, a statistic that has not changed in almost 50 years [
1]. Until recently, gemcitabine was the current standard for post-operative chemotherapy, delaying the development of recurrent disease in some PDAC patients, with a modest improvement in overall survival achieved upon combination with the epidermal growth factor receptor (EGFR) -directed tyrosine kinase inhibitor erlotinib [
2]. However, recent studies indicate that the addition of nab-paclitaxel to gemcitabine can provide a significant survival benefit [
2], and the combined chemotherapeutic modality Folfirinox has emerged as a more effective treatment than gemcitabine, although at the cost of significant toxicity [
2]. Further characterization of the molecular pathways regulating PDAC development and progression may lead to the identification of improved therapeutic strategies, as well as biomarkers that help stratify patients for optimal treatment.
Multiple lines of evidence support a role for deregulated tyrosine kinase signaling in PDAC development and progression. For example, activating KRas mutations represent the earliest known genetic alteration in PDAC [
3], and evidence from genetically-modified mouse models support their functional role in this malignancy [
4]. Signaling by the EGFR is required for acinar-ductal metaplasia (ADM), an early step in PDAC progression, as well as survival of cells in established ADM and pancreatic intraepithelial neoplasia lesions [
5], and overexpression of the EGFR [
6] and the related receptor ERBB3 [
7] has been detected in PDAC, with aberrant expression being associated with poor prognosis. Alterations can also occur in downstream signaling components. For example, AKT2 gene amplification [
8], and loss of the tumour suppressor PTEN [
9], occur in this disease. Furthermore, tyrosine phosphorylation of the transcriptional regulator and downstream target of JAK signaling, Stat3, is enhanced in PDAC compared to normal tissue [
10], and is associated with poor outcome in resected disease [
11]. Importantly, conditional ablation of Stat3 in KRas-driven mouse models of PDAC have confirmed the importance of Stat3 signaling in ADM [
12], pancreatic intraepithelial neoplasia (panIN) formation [
12,
13] and panIN progression and PDAC development [
12,
14].
SgK223/Pragmin [
15], and SgK269/PEAK1 [
16] are large, closely-related proteins that possess a kinase-like domain at their C-termini. However, these kinase-like domains contain substitutions in key motifs characteristic of
bona fide kinases, such as the DFG motif responsible for Mg
2+-ATP binding, where the aspartate residue is substituted by asparagine. Since both proteins lack nucleotide binding activity based on a thermal shift assay, they likely represent pseudokinases [
17]. N-terminal to the pseudokinase domain, both proteins contain tyrosine phosphorylation sites that recruit specific SH2 and PTB domain-containing effectors, indicating that SgK223 and SgK269 undertake a scaffolding role during tyrosine kinase signaling. For example, SgK223 binds to Csk, a negative regulator of Src, via SgK223 Y411 [
18], while SgK269 binds to Grb2 and Shc1 via Y635 and Y1188 to promote proliferative and morphogenic signals, respectively [
19,
20]. Recent work has determined that SgK269 plays a key role during growth factor receptor signaling, mediating a qualitative switch in EGFR output from proliferative/survival signaling to promotion of cell migration/invasion [
20].
Importantly, SgK223 and SgK269 both exhibit emerging oncogenic roles. For example, SgK223 promotes cell invasion in colon carcinoma cells exhibiting high Src activity [
21], while overexpression of SgK269 promotes growth and aberrant morphogenesis of MCF-10A mammary epithelial cells, and is required for epithelial-to-mesenchymal transition (EMT) and anchorage-independent growth of basal breast cancer cells [
19]. In addition, SgK269 is required for efficient tumour formation and metastasis in an orthotopic pancreatic cancer xenograft model [
22]. SgK269 is overexpressed in colon, pancreatic and breast cancers relative to normal tissue [
19,
22,
16], but the expression profile of SgK223 in human malignancies is poorly characterized.
In this study we demonstrate that SgK223 exhibits enhanced phosphorylation and/or expression in PDAC cell lines and tumours relative to normal controls. In addition, we identify a novel pathway linking SgK223, Stat3 and an invasive phenotype during PDAC development. Overall this work provides important new insights into the signaling and oncogenic function of this pseudokinase scaffold.
Discussion
Several studies have implicated Stat3 activation in PDAC development and progression [
12,
11,
13,
14,
10]. Previous reports indicated that the major signaling pathway leading to activation of Stat3 in PDAC is cytokine-induced tyrosine phosphorylation of the signaling co-receptor gp130 and JAK activation [
12], and this can occur in a cell autonomous fashion [
12,
13], as well as via a cell non-autonomous mechanism involving IL-6 production by infiltrating myeloid cells [
14]. Increased production and/or exposure to cytokines, and upregulation of gp130, represent identified mechanisms for the enhanced activation of Stat3 detected in PDAC versus normal tissue [
12‐
14]. Our identification of increased expression of SgK223 in PDAC versus normal tissue, and demonstration that SgK223 is sufficient to increase Stat3 activation in HPDE cells and is required for Stat3 phosphorylation in AsPC-1 PDAC cells, now identify aberrant expression of this pseudokinase as an additional mechanism for enhancement of Stat3 signaling in PDAC. While all details of SgK223 action on this pathway have yet to be resolved, our work identifies certain key aspects. First, since JAK1 activation was enhanced in SgK223-overexpressing cells, and selective JAK inhibitors could normalize Stat3 phosphorylation, SgK223 must act upstream of JAK1 to amplify Stat3 signaling. Second, increased production of IL6, and altered expression of gp130, do not appear to be involved. Third, since SgK223 associates with Stat3, this implicates the scaffolding function of SgK223 in regulation of Stat3 phosphorylation, although SgK223 does not itself ‘bridge’ Stat3 and JAK1. The possibility that SgK223 complexes both gp130 and Stat3, or alters the activity of particular protein tyrosine phosphatases, such as TC-PTP [
26], towards Stat3, is currently under investigation. Whatever the mechanism, the phosphorylated Stat3 generated in SgK223-overexpressing cells is competent for nuclear translocation and transcriptional activation of target genes, as confirmed by our reporter assays.
Reflecting their relationship as paralogues, SgK223 and SgK269 exhibit similarities and differences in function and signal output. Both proteins regulate cell morphology, promoting a more elongated phenotype [
19,
18] and a partial EMT [
19]. With regard to the latter process, it is noteworthy that increased expression of either protein led to enhanced expression of MITF, a transcription factor that regulates the balance between migration/invasion and proliferation in melanoma cells, and particular TGFβ family members, that are known to promote acquisition of a mesenchymal phenotype (Fig.
2) [
19,
27,
28]. In addition, both SgK223 and SgK269 modulate the tyrosine phosphorylation of specific erbB receptors. The Klemke group [
22] demonstrated that SgK269 associated with erbB2, and SgK269 knockdown decreased phosphorylation of this receptor on specific sites, while we observed enhanced EGFR tyrosine phosphorylation upon SgK223 overexpression. However, while SgK269 enhanced site-selective tyrosine phosphorylation of paxillin and p130Cas [
16], this direct impact on focal adhesion signaling was not observed in our SgK223 overexpression model. In addition, while SgK269 increases Erk activation [
19,
16], this was not observed in HPDE cells overexpressing SgK223 (Fig.
3). This likely reflects the absence of a direct Grb2 binding site in SgK223, while SgK269 recruits this adaptor via Y635 [
19].
Interestingly, previous studies reported positive regulation of Src by SgK223 and SgK269 [
22,
18]. In the case of the former pseudokinase, this reflected sequestration of Csk in the cytosol by SgK223, leading to activation of plasma membrane-localized Src [
18]. However, this was demonstrated using transient expression assays in gastric epithelial cells, and in our HPDE cells stably expressing SgK223, Src activity was decreased, likely reflecting a robust increase in Csk expression in the SgK223-overexpressing cells, which may represent a negative feedback mechanism. Taking these data and those from the Hatakeyama group in combination, the effect of SgK223 on Src may be context-dependent, and vary according to the relative abundance of Src and its regulators in specific subcellular localizations.
An additional conserved feature of SgK223 and SgK269 is their ability to enhance activation of Stat3 ([
19], this paper). In the case of SgK223, we utilized the Stat3 inhibitor Stattic to demonstrate that a key role for SgK223-induced Stat3 signaling is enhanced cellular migration and invasion. The cellular function of Stat3 signaling downstream of SgK269 has yet to be defined, although mutation of Y635 to phenylalanine, which abrogates SgK269-mediated activation of both Erk and Stat3, negatively impacts upon both proliferation in 3D culture and cellular invasion [
19]. Consequently, it appears likely that Stat3 represents a common effector utilized by both pseudokinases to drive acquisition of a migratory and invasive phenotype. This highlights an important oncogenic role for both SgK223 and SgK269 in PDAC, given that both are overexpressed in this malignancy (this paper, [
22]) and the critical role played by Stat3 in PDAC development [
12‐
14] and metastasis [
29]. In addition, the strong links between these pseudokinases and Stat3 signaling indicate that SgK223 and SgK269 represent candidate biomarkers for responsiveness to targeted therapies directed at the JAK/Stat3 pathway.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors of this manuscript have no conflicts of interest to declare.
Authors’ contributions
CMT characterised SgK223 expression in PDAC cell lines, manipulated SgK223 expression and undertook cellular and signaling assays, and drafted the manuscript. YWP, LL and LZ undertook additional experimentation involving cell signalling assays and phosphoproteomic analysis on the HPDE/SgK223 cells. ESH performed the phosphoproteomic profiling across the PDAC panel and analysed the resulting data. MC and MP analysed the gene expression data and undertook statistical analyses. RJD conceived of the study, participated in its design and coordination and finalized writing of the manuscript. AVB contributed to project co-ordination. All authors read and approved the final manuscript.