Pancreatic stellate cell secreted IL-6 stimulates STAT3 dependent invasiveness of pancreatic intraepithelial neoplasia and cancer cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a dynamic tumor supported by several stromal elements such as pancreatic stellate cells (PSC). Significant crosstalk exists between PSCs and tumor cells to stimulate oncogenic signaling and malignant progression of PDAC. However, how PSCs activate intercellular signaling in PDAC cells remains to be elucidated. We have previously shown that activated signal transducer and activator of transcription 3 (STAT3) signaling is a key component in the progression of pancreatic neoplasia. We hypothesize that PSC secreted IL-6 activates STAT3 signaling to promote PanIN progression to PDAC. Human PDAC and mouse PanIN cells were treated with PSC-conditioned media (PSC-CM), and phospho- and total-STAT3 levels by immunoblot analysis were determined. IL-6 was quantified in PSC-CM and cell invasion and colony formation assays were performed in the presence or absence of a neutralizing IL-6 antibody and the JAK/STAT3 inhibitor AZD1480. Serum from Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) and LSL-KrasG12D/+; Trp53R172H/+; Pdx1Cre/+ (KPC) mice demonstrated increased levels of IL-6 compared to serum from non-PDAC bearing KC and PK mice. PSC secreted IL-6 activated STAT3 signaling in noninvasive, precursor PanIN cells as well as PDAC cells, resulting in enhanced cell invasion and colony formation in both cell types. There was a significant positive linear correlation between IL-6 concentration and the ratio of phosphorylated STAT3/total STAT3. IL-6 neutralization or STAT3 inhibition attenuated PSC-CM induced activation of STAT3 signaling and tumorigenicity. These data provide evidence that PSCs are directly involved in promoting the progression of PanINs towards invasive carcinoma. This study demonstrates a novel role of PSC secreted IL-6 in transitioning noninvasive pancreatic precursor cells into invasive PDAC through the activation of STAT3 signaling.

Keywords: IL-6; PanIN; STAT3; pancreatic cancer; pancreatic stellate cells.

Figure 1. Pancreatic stellate cell (PSC) conditioned media (CM) activates STAT3

A. Baseline STAT3 level was obtained by immunoblotting total cell lysates from human (left panel) PSC (hPSCs), pancreatic ductal cells (H6c7), and PDAC cells lines (BxPC3, PANC1), as well as mouse (right panel) PSC (mPSCs), PanIN, PDA and liver metastasis (LMP) cell lines. Only the tumor cell lines expressed high levels of pSTAT3 at baseline. Densitometry analyses for the immunoblots from pSTAT3 (top panel) and tSTAT3 (bottom panel) after normalization to actin protein is shown to the right. B and C. Serial exposure to concentrated mPSC-CM results in activation of STAT3 in mouse PanIN cells in a dose- (B) and time- (C) dependent manner. Densitometry analyses of (B) and (C) pSTAT3 immunoblots normalized to tSTAT3 are shown in the graphs to the right. D. PANC1 and MiaPaCa2 cells also demonstrate a dose- dependent increase in STAT3 activation when exposed to hPSC-CM (left panel). Densitometry analyses of pSTAT3 immunoblots normalized to tSTAT3 for PANC1 (top panel) and MiaPaCa2 (bottom panel) cells are displayed in graphs to the right.

Figure 2. IL-6 concentration in mouse and human PSC-CM

Levels of IL-6 were measured by ELISA relative to total protein levels in serially diluted CM secreted from mPSCs A. and hPSCs B. Inset for hPSCs (B) shows the same concentration range as provided for the mPSCs (A). The Relationship between recombinant IL-6 concentration and STAT3 activation; C. The ratio of pSTAT3 to tSTAT3 levels in mouse PanIN cells was determined by quantifying pSTAT3 and tSTAT3 luminescence on the immunoblots from Figure 1B (ImageJ) and performing a Pearson's correlation (p < 0.001). D. The relationship between IL-6 concentration and ratio of pSTAT3 to tSTAT3 levels in human MiaPaCa2 cells was determined by quantifying pSTAT3 and tSTAT3 luminescence on the immunoblots from Figure 1C (ImageJ) and performing a Spearman's correlation (p < 0.001). E. and F. In vivo analysis of IL-6 from the serum collected from LSL-Kras^G12D/+; Pdx1^Cre/+ (KC) and LSL-Kras^G12D/+; Trp53^R172H/+; Pdx1^Cre/+ (KPC) mice (E) or Ptf1a^Cre/+; LSL-Kras^G12D/+ (PK) and Ptf1a^Cre/+; LSL-Kras^G12D/+; Tgfbr2^flox/flox (PKT) mice (F). Serum from 3 mice was analyzed in triplicates (n=9). * – p<0.05; *** – P<0.001.

Figure 3. Pharmacological inhibition of JAK/STAT3 signaling or blocking IL-6 inhibits phosphorylation of STAT3 in hPSC-CM protein PDAC treated cells

PANC1 and BxPC3 cells were treated with hPSC-CM with or without JAK/STAT3 inhibitor AZD1480 (100 nmol/L) A. or IL-6 neutralizing antibody B. At the end of the study, cell lysates were analyzed for total STAT3 and phospho-STAT3 levels by immunoblot analysis. Densitometry analyses of pSTAT3 normalized to tSTAT3 was shown in the bottom panels of A and B. AZD1480 or IL-6 Ab treatment inhibited hPSC-CM induced activation of STAT3.

Figure 4. Blocking IL-6 attenuates hPSC-CM induced cell invasion and colony size

PANC1 cells were treated with hPSC-CM (100μg total protein/ml) with or without IL-6 neutralizing antibody (IL-6 Ab). Cell invasion A. and colony size B. were analyzed as detailed in Materials and Methods. Cell invasion images demonstrate representative pictures from each treatment group (A, right panel). Cell invasion results represent the average number of cells in seven fields in triplicate inserts. Colony size results are represented by eight photographs analyzed from triplicate wells. * – P < 0.05.

Figure 5. Mouse PSC-CM mediates activation of PanIN cell invasion through IL6-JAK-STAT3 signaling

Mouse PanIN cells were treated with mPSC-CM (100μg total protein/ml) with or without AZD1480 (100 nmol/L) or IL-6 neutralizing Ab and analyzed for cell invasion. A. Images demonstrate representative pictures from each treatment group. B. PanIN cell invasion analysis results showed that either IL-6 neutralizing Ab or AZD1480 significantly blocked mPSC-CM induced activation of cell invasion. * – P < 0.05; ** – P < 0.01.