Despite significant advances in the understanding of cancer biology during recent decades, pancreatic cancer remains one of the deadliest types of human cancer
[1–3]. Since the introduction of gemcitabine in 1996, which is currently the gold standard for the treatment of advanced pancreatic cancer, only the EGFR TKI erlotinib has gained FDA approval for the treatment of patients with metastatic pancreatic cancer in combination with gemcitabine
. This combination resulted in a modest, but statistically significant survival benefit however, many patients simply do not respond or acquire resistance following a short course of therapy
[25, 26]. Recent studies have demonstrated that IGF-IR is implicated in resistance to anti-HER targeted therapy and that simultaneous targeting of both IGF-IR and EGFR or IGF-IR and HER-2 may lead to a superior therapeutic effect compared to treatment with the single agent in breast and glioblastoma, prostate and colorectal cancer cells
To date, the number of studies investigating the effect of IGF-IR inhibitor NVP-AEW541, in pancreatic cancer is limited
[37–39]. To the best of our knowledge this is the first study investigating the therapeutic potential of this approach in pancreatic cancer using a pan-HER bocker (afatinib) and IGF-IR TKI NVP-AEW541. We have reported recently the superiority of afatinib compared to our anti-EGFR mAb ICR62 and erlotinib in inhibiting the growth of a panel of human pancreatic cancer cell lines. As a single agent, afatinib inhibited the growth of all pancreatic cancer cell lines with IC50 values ranging from 11 nM (BxPC-3) to 1.37 μM (FA6)
. Interestingly, BxPC-3, which is the only one carrying a wild-type K-Ras gene, was the most sensitive cell line to treatment with HER inhibitors
. In addition, we found that treatment with a combination of afatinib and gemcitabine resulted in the synergistic growth inhibition of the majority of human pancreatic cancer cells (BxPC-3, AsPc-1, FA6, PANC-1 and Capan-1)
. In this study, we investigated the sensitivity of the same panel of pancreatic cancer cells to treatment with NVP-AEW541 when used alone or in combination with gemcitabine, ICR62 or afatinib. We found NVP-AEW541 to inhibit the growth of all pancreatic cancer cell lines with IC50 values ranging from 342 nM (FA6) to 2.73 μM (PT-45) (Figure
1). Western blot analysis revealed that, NVP-AEW541 inhibited completely the ligand-induced phosphorylation of IGF-IR and AKT in FA6 but not in the more resistant BxPC3 cells (IC50= 1.54 μM) (Table
6). We also investigated the growth response of these cancer cell lines to treatment with PI3K and MAPKK inhibitors and found that these were less effective compared to afatinib and NVP-AEW541 (Figure
1). Since the IC50 values of these inhibitors for their respective targets are below 2 μM (0.07 μM for MAPKK inhibitor, 1.4 μM for PI3K inhibitor), our results suggest that the panel of pancreatic cancer cell lines used in this study is highly resistant to inhibition of PI3K and MAPKK.
We next assessed the anti-tumour activity of these agents when used in combination. There was no improvement in anti-tumour activity when NVP-AEW541 was used in combination with mAb ICR62 (data not shown). Treatment with a combination of gemcitabine and NVP-AEW541 resulted in synergistic growth inhibition only in PANC1 cell line (Table
2). Interestingly, treatment with a combination of NVP-AEW541 and afatinib was found to be superior, leading to a synergistic growth inhibition of all pancreatic cancer cells with the exception of PT45 which was the most resistance cell line to treatment with NVP-AEW541 (Table
2). Synergism following treatment with a combination of NVP-AEW541 and HER inhibitors (e.g. trastuzumab, erlotinib) has previously been reported in studies involving breast and colorectal cancer cells
[36, 40, 41].
Investigation of the effect of IGF-IR ligands (IGF-I, IGF-II and Insulin) and HER ligands EGF and NRG-1 on the downstream signaling in BxPc3 cells revealed that EGF primarily induces phosphorylation of MAPK while IGF-IR ligands activate predominantly the PI3K/AKT pathway. The activation of different pathways by the HER family and IGF-IR systems could explain the synergistic effect exhibited by the combination of pan-HER blocker afatinib and IGF-IR inhibitor in this cell line. In optimal growth conditions (10% FBS supplemented medium) afatinib was more potent at down regulating both AKT and MAPK basal phosphorylation levels while NVP-AEW541 downregulated pAKT but had no effect on pMAPK basal levels in BxPc3 cells. However, even though afatinib was more effective at downregulating pAKT than NVP-AEW541, only the combination of NVP-AEW541 with afatinib led to complete loss of AKT phosphorylation (Figure
In order to determine whether the diverse activation of AKT and MAPK pathways by EGFR and IGF-IR activation could explain the synergism exhibited by the same combination in the rest of the cell lines we determined the effect of EGF and IGF on the phosphorylation of AKT and MAPK in all cell lines included in this study. Interestingly, with the exception of FA6 cells, the pattern of AKT and MAPK activation in all the other pancreatic cells was found to be similar to BxPc3 cells (Figure
5); EGF predominantly led to the activation of MAPK whereas IGF treatment increased mainly the phosphorylation of AKT but had low or no effect on phosphorylation of MAPK. This in turn suggests that the synergistic effect by this combination may be driven by more effective and simultaneous blockade of HER family members and IGF-IR induced phosphorylation of both AKT and MAPK. However, further studies investigating the effect of this combination in other signaling pathways such as the JAK-STAT pathway, and the effect of the mutational status of downstream cell signalling molecules (e.g. IRS, PTEN and K-ras), on the synergistic potential of this combination, are necessary in order to elucidate the exact mechanism involved in the synergism observed by this combination.
All of the pancreatic cancer cell lines examined in this study were found to be IGF-IR positive, and in the majority of the cases, the expression levels were similar to that of the IGF-IR positive MCF-7 control cell line consequently, there was no correlation between IGF-IR expression and response to treatment with NVP-AEW541, indicating that additional factors are implicated in the sensitivity of these cell lines to IGF-IR inhibition (Table
1). Lack of any clear association between IGF-IR expression and response to NVP-AEW541 has also been found in previous studies investigating the effect of this agent against colorectal and breast cancer cell lines
In order to investigate the dependency of each cell line to the HER and IGF-IR signalling pathways, we determined the growth response of these cell lines to several exogenous HER and IGF-IR ligands. Results showed that while the majority of cell lines did not respond to treatment with exogenous HER ligands, several cell lines demonstrated increased growth following treatment with IGF-IR ligands (BxPc3, AsPc-1, Capan-1 and PT45) indicating that IGF-IR may have a more important biological role in this panel of pancreatic cancer cell lines. In addition, the fact that two cell lines (AsPc-1 and Capan-1) responded to some HER ligands but not others (e.g. AsPc-1 responded to epigen treatment only but not to any other EGFR ligand) indicates that different ligands can have a diverse impact on the proliferation of each pancreatic cancer cell line (Figure
2). Furthermore, our results suggest that there is no correlation between growth response to these exogenous ligands and inhibition of their respective receptors. For example, FA6 cell line which exhibited the highest sensitivity to IGF-IR inhibition (IC50 = 342 nM) by TKI NVP-AEW541, was growth stimulated by 5-7% following treatment with either IGF-I, IGF-II or insulin. In contrast, BxPc3, which is a more resistant cell line to IGF-IR inhibition (IC50 = 1.54 μM), exhibited more than 30% increase in growth following treatment with the same ligands (Figure
2). Therefore, other factors such as the level of autocrine ligands, the expression and status of downstream cell signalling molecules, as well as the level of cross-talk between different RTKs may influence sensitivity to IGF-IR inhibition
Several studies investigating the therapeutic potential of IGF-IR inhibition have been met with disappointing results, indicating that the potential of this receptor as a single target may be rather limited
. Interestingly, our results show that NVP-AEW541 is effective at inhibiting the growth of human pancreatic tumour cells and that the combination of NVP-AEW541 and afatinib is superior in terms of synergistic effect to the combination of either agent with gemcitabine. Taken together, our findings encourage further investigation in vivo on the therapeutic potential of this combination in pancreatic cancer.