Targeting ErbB3-mediated stromal-epithelial interactions in pancreatic ductal adenocarcinoma

Abstract

Background: We sought to investigate the role of ErbB3-mediated signalling on the interaction between pancreatic cancer-associated fibroblasts (CAF) and carcinoma cells in an effort to disrupt tumourigenic pancreatic ductal adenocarcinoma (PDAC) stromal-epithelial cross-communication.

Methods: Primary CAF cultures were established from human PDAC surgical specimens. AsPC-1 pancreatic cancer cell murine subcutaneous xenografts were developed in the presence and absence of CAF and were subsequently treated with epidermal growth factor receptor (EGFR) inhibitors (erlotinib) and ErbB3 inhibitors (MM-121, monoclonal ErbB3 antibody).

Results: Cancer-associated fibroblasts were found to secrete neuregulin-1 (NRG-1), which promoted proliferation via phosphorylation of ErbB3 and AKT in AsPC-1 PDAC cells. This signalling cascade was effectively inhibited both in vitro and in vivo by specific ErbB3 blockade with MM-121, with greater degree of tumourigenesis inhibition when combined with erlotinib. The CAF-AsPC-1 pancreatic cancer xenografts reached significantly greater tumour volume than those xenografts lacking CAF and were resistant to the anti-tumour effects of EGFR inhibition with erlotinib.

Conclusion: Cancer-associated fibroblasts-derived NRG-1 promote PDAC tumourigenesis via ErbB3-AKT signalling and overcomes single-agent EGFR inhibition. Disruption of this stromally mediated tumourigenic mechanism is best obtained through combined EGFR-ErbB3 inhibition with both erlotinib and MM-121. We have identified the NRG-1/ErbB3 axis as an attractive molecular target for the interruption of tumourigenic stromal-epithelial interactions within the PDAC microenvironment.

Figure 1

(A) Ligand expression differences between stroma and tumour in microdissected pancreatic cancer samples. ΔC_t values were calculated as a difference between the target gene C_t and RPLPO (housekeeping gene) C_t values (C_t=threshold cycle). Bars represent mean values±s.e.m. P=P-value for Mann–Whitney rank test. (B) IHC analysis of two primary CAF cultures CAF-1 and CAF-4 together with Panc-1 and BxPC-3 cell lines. (C) Gene expression differences between CAF and normal tissue fibroblasts. ΔC_t values are calculated against RPLPO C_t values. Bars represent mean values±s.e.m. P=P-value for two-tailed t-test.

Figure 2

Western blot analysis of NRG-1 secretion into CAF-conditioned media filtered with 30 kDa cutoff filter (both fresh and after a single freeze-thaw cycle).

Figure 3

Western blot and proliferation analysis of CAF-conditioned media effects on AsPC-1 cells. Addition of ErbB3 blocking peptide or anti-NRG-1 neutralising antibody partially abrogates the stimulating effect of CAF-CM.

Figure 4

NRG-1β abrogates the inhibitory effect of erlotinib in vitro. (A) ErbB3 expression analysis of nine representative pancreatic cell lines. (B) Relative proliferation of nine pancreatic cell lines treated with erlotinib with subsequent stimulation with either EGF or NRG-1β. Proliferation of the erlotinib-treated control cells is set at 1.0 on the y axis.

Figure 5

Addition of neutralising NRG-1 Ab limits proliferative escape from erlotinib-induced inhibition in the presence of CAF-conditioned media.

Figure 6

AsPC-1 tumour growth without CAF (•) and with CAF at 1 : 1 ratio (▪) and 1 : 2 ratio (▴).

Figure 7

Effects of CAF on tumourigenesis and influence of erlotinib treatment on AKT activation. (A) Tumour volume difference between AsPC-1+CAF and AsPC-1 alone xenogratfs on day 13 post-inoculation. (B) Comparison of relative tumour growth between four groups of mice. All data points are represented by mean values with bars showing s.e.m. (C) Western blot analysis of AKT activation in eight representative tumours from each of the four groups of mice. Numbers underneath each lane represent the absolute band intensity.

Figure 8

IHC analysis of xenograft tumours with and without CAF and effect of erlotinib treatment. The photographs (A, B) show low power (4 × ) representative sections of α-SMA staining of AsPC-1 and AsPC-1+CAF xenografts, respectively. The photographs (C, D) show representative sections of pErbB3 expression in erlotinib-treated AsPC-1 and AsPC-1+CAF xenografts. A high power (20 × ) view demonstrates positive membranous pattern, scored as intensity 2 on a 0–3 scale (D), the opposite (C) shows negative staining. The photographs (E, F) show representative sections of pErbB3 staining in placebo-treated AsPC-1 and AsPC-1+CAF xenografts.

Figure 9

Effects of MM-121 ErbB3 antibody on AsPC-1 cell proliferation and signalling both in vitro and in vivo. (A) Western blot analysis of inhibitory effect of erlotinib, MM-121 and their combination following EGF or NRG-1 stimulation. (B) Inhibition of AsPC-1 proliferation with erlotinib, MM-121 and their combination. (C) MM-121 inhibits AsPC-1 tumour progression in a dose-dependent manner. (D) Western blot analysis of AsPC-1 xenografts depicting the inhibition of ErbB3 activation and expression and inhibition of pAKT.