Complement C3 overexpression activates JAK2/STAT3 pathway and correlates with gastric cancer progression

Abstract

Background: Localized C3 deposition is a well-known factor of inflammation. However, its role in oncoprogression of gastric cancer (GC) remains obscured. This study aims to explore the prognostic value of C3 deposition and to elucidate the mechanism of C3-related oncoprogression for GC.

Methods: From August to December 2013, 106 GC patients were prospectively included. The regional expression of C3 and other effectors in gastric tissues were detected by WB, IHC, qRT-PCR and other tests. The correlation of localized C3 deposition and oncologic outcomes was determined by 5-year survival significance. Human GC and normal epithelial cell lines were employed to detect a relationship between C3 and STAT3 signaling pathway in vitro experiments.

Results: C3 and C3a expression were markedly enhanced in GC tissues at both mRNA and protein levels compared with those in paired nontumorous tissues. According to IHC C3 score, 65 (61.3%) and 41 (38.7%) patients had high and low C3 deposition, respectively. C3 deposition was negatively correlated with plasma levels of C3 and C3a (both P < 0.001) and positively correlated with pathological T and TNM stages (both P < 0.001). High C3 deposition was identified as an independent prognostic factor of poor 5-year overall survival (P = 0.045). In vitro C3 administration remarkably enhanced p-JAK2/p-STAT3 expression in GC cell lines but caused a reduction of such activation when pre-incubated with a C3 blocker. Importantly, C3 failed to activate such signaling in cells pre-treated with a JAK2 inhibitor.

Conclusions: Localized C3 deposition in the tumor microenvironment is a relevant immune signature for predicting prognosis of GC. It may aberrantly activate JAK2/STAT3 pathway allowing oncoprogression.

Trial registration: ClinicalTrials.gov, NCT02425930, Registered 1st August 2013.

Keywords: Complement; Gastric cancer; Immune signature; Prognosis prediction; STAT3; Tumor stage.

Conflict of interest statement

All authors report no conflicts of interest in this work.

Figures

Fig. 1

The flow chart of clinical study. Patients qualified with our study criteria were prospectively enrolled, with written informed consent obtained before any treatment. Included patients were allocated to two groups based on the average IHC C3 score

Fig. 2

Increased expression of C3 in GC tissues. a Analysis of C3 expression in unpaired GC tissues and normal tissues in TCGA cohort (P < 0.001, left panel), C3 expression in paired GC and adjacent normal tissues (n = 37) in TCGA cohort (P < 0.001, middle panel), with C5 expression in the paired cohort (P = 0.546, right panel); b C3 expression in Gastric tissues according to Oncomine dataset (P = 2.49E-5; Reporter ID: 217767_at); c The protein levels of complement C3 and its effectors detected with western blot method in GC tissues and respective adjacent normal tissues (left panel; n = 3, left panel), with relative protein levels of C3 and C3a (right panel; n = 106, P < 0.001 vs normal tissues); Deposition of C3, C3a, C5a and the presence of CR1 and factor B in GC tissues were measured with IHC (d) and IFC (e) staining methods, with normal tissues utilized as control. Representative images of n = 5 independent experiments

Fig. 3

C3 deposition in GC tissues was associated with systemic Complement depletion. Regional deposition of C3 in GC tissues can be evaluated by the intensity of IHC staining (a), with score 0–3 for negative, weak, moderate and strong staining respectively. The average score of C3 deposition was calculated by five independent high-power fields of IHC section from each patient. The average score in primary GC tissues was much higher than that in adjacent normal tissues (b, left panel). Additionally, the case distribution based on C3 score presented as low (n = 41) and high C3 deposition (n = 35 as high and n = 30 as severe) in GC tissues (b right panel). The filling colors were black, brown and red for low, high and severe IHC scores, respectively. Linear relationship between average IHC C3 score and plasma levels of C3 and C4 at baseline and surgery was investigated (c). The relationship between IHC score and intraoperative plasma levels of C3a (P < 0.001), fB (P < 0.001) and C5a (P = 0.444) was explored using ELISA method (d, n = 30). The mRNA expression of C3 in GC tissues and C5 in the peripheral blood were extracted from TCGA database to assess their relationship, with no correlation found (e; P = 0.137, n = 384)

Fig. 4

Enhanced deposition of C3 in GC tissues predicts advanced tumor stage and poor prognosis. a Regional deposition of C3 in GC tissues was strongly associated with advanced T stage and TNM stage (P < 0.001); however, it was not correlated with pathological N stage and clinical TNM stage in the current GC cohort (P > 0.05); b The 5-year overall survival and disease-free survival curves based on C3 deposition in enrolled subjects and TCGA samples; c The overall survival differences according to various tumor stage in our dataset; d The ROC curves of oncologic outcome (cancer-related death) based on C3 deposition, baseline C3 depletion, pathologic TNM stage, tumor markers (CEA and CA19–9) and combined factors (C3 deposition plus CEA); e Forest plot of short-term surgical outcomes (postoperative morbidities), with relative risk (RR) compared between the two groups

Fig. 5

The long-term recurrence-free survival of GC patients with low or high C3 deposition in each tumor stage. Kaplan-Meier curves were employed to compare survival significance among stage I, II and III. Of note, patients with stage IV were excluded from such comparison

Fig. 6

Enhanced expression of C3 promoted tumor progression in GC cell lines. a Overexpression of C3 in human GC cell lines (SGC-7901 and MGC-803) detected by western blot and qRT-PCR methods, with normal gastric cell line (GES-1) as control; b Exogenous C3 stimulation promoted the migration of GC cells (left panel). The time-dependent cell proliferation was inhibited by CVF in both GC cell lines (right panel); c Inhibition of C3 activation with CVF significantly inhibited the invasion of GC cells; d Flow cytometry study to investigate the apoptosis rate of GC cells. Early stage of apoptosis was detected by propidium iodide (PI) and annexin V-fluorescein isothiocyanate (V-FITC) dual staining assay. 20,000 cells per sample in all in vitro assays, representative sparklines and histograms (right panel) of n = 5 independent experiments

Fig. 7

JAK2/STAT3 signaling pathway was related to C3 deposition in GC tissues and C3-induced oncoprogression. a Typical expression of p-STAT3 and IL-6 in GC and adjacent normal tissues (IFC method), which indicates an up-regulation of STAT3 signaling in GC patients (representative images of n = 5 independent experiments); b Levels of JAK2/STAT3-related proteins (IL-6, p-JAK2, p-STAT3 and STAT3) were detected on SGC-7901 and normal GES-1 cell line with WB method (left panel). The STAT3 signaling was highly activated with exogenous C3 treatment, and greatly inhibited when JAK2-blocker (AG490) was pre-incubated with C3 (right panel); c Levels of p-STAT3 and IL-6 in C3-antagonist pre-treated GC cells (upper panel). The JAK2/STAT3 signaling remained activated but weakened under blockage of C3 signaling with CR1 compared with blockage of JAK2 with AG490 (lower panel). 20,000 cells per sample in all in vitro assays, representative histograms (right panel) of n = 5 independent experiments; d A proposed model for the underlying mechanism of C3/JAK2/STAT3 signaling pathway participating in the pathogenesis of GC. Abbreviations: 3aR, Complement C3a receptor; MAC, membrane attack complex; CVF, cobra venom factor