So far, gemcitabine has demonstrated disappointing results in patients with pancreatic cancer. More effective chemotherapeutics like FOLFIRINOX, where chemoresistance also remains a major clinical problem, is associated with frequent dose reductions, treatment-related serious adverse events, and often grade 3-4 infections. Since gemcitabine is much better tolerated with less toxicity compared to the newer chemotherapeutic agents, especially in the elderly pancreatic cancer patients, we aimed to evaluate whether the anti-tumor effect of gemcitabine could be enhanced by IFN-β and to demonstrate the mechanism of action.
First, we studied the effect of IFN-β alone and in combination with gemcitabine in three human pancreatic cancer cell lines: two IFN-β sensitive cell lines (BxPC-3 and CFPAC-1) and an IFN-β insensitive cell line (Panc-1).(16)
IFN-β strongly increased the inhibitory effects of gemcitabine in the IFN-β sensitive cell lines, which was confirmed in colony-forming assay. Both the cytotoxic and cytostatic effects of gemcitabine were significantly enhanced by IFN-β.
The increased chemosensitivity can be explained by two important observations. First, IFN-β increased cell population in the S-phase, suggesting that these cells were not able to transit into the G2/M-phase efficiently, and therefore exhibited a prolonged stay in the S-phase. As a result, DNA replication fails and cells can become more vulnerable for gemcitabine treatment. A downregulation and impaired activity of cyclins and cyclin dependent kinases was previously reported after IFN-β treatment, explaining the prolonged stay in the S-phase.(23) The active metabolites of gemcitabine, dFdCDP and dFdCTP, are also known to inhibit DNA synthesis by inhibiting ribonucleotide reductase and by DNA incorporation, respectively. Consequently, a complete inhibition of DNA synthesis is achieved, and apoptosis is induced.(7) Combination therapy with IFN-β and gemcitabine resulted in the strongest induction of cells in the S-phase in the IFN-β sensitive cell lines. In Panc-1, no difference was observed upon IFN-β treatment, which is in line with the absence of the chemosensitising effect in this cell line.
The second observation is the upregulation of gemcitabine transporters by IFN-β, resulting in potentially increased drug influx. A strong correlation was found between treatment resistance and the expression of gemcitabine transporters (hENT1, hCNT1, and hCNT3), activating enzyme (dCK), and inactivating enzyme (CDA) of gemcitabine.(6, 7) It should be emphasized, however, that we studied the effect of IFN-β on mRNA expression of gemcitabine transporters only. Further studies are necessary to demonstrate that IFN-β treatment results in increased intracellular gemcitabine levels and to confirm this mechanism of action by e.g. transporter knockdown. Nevertheless, this is the first study that evaluated the potential interaction between IFN-β and the expression of genes involved in gemcitabine transport and metabolism of gemcitabine. IFN-β strongly increased the expression of genes encoding for the hCNT1 and hCNT3 transporter in BxPC-3 and CFPAC-1. In contrast, IFN-β upregulated expression of CDA in Panc-1 cells.
The time of incubation with IFN-β appeared to be a significant parameter in our study. First, the (chemosensitising) anti-tumor effect was time-dependent. Notably, a 4 hr pre-treatment with IFN-β already increased the response to gemcitabine in the IFN-β sensitive cell lines. In line with this, expression of ISGs was upregulated after 4 hr and increased over time. Secondly, IFN-β did not enhance the gemcitabine response when both drugs were given simultaneously, suggesting that IFN-β needs time to sensitize tumor cells for chemotherapy.
Remarkably, there were significant differences between the IFN-β sensitive cell lines BxPC-3 and CFPAC-1 and the relative insensitive cell line Panc-1. First, the anti-tumor effects of IFN-β were less pronounced in Panc-1, even though these cells were treated with a 10-fold higher concentration (1000 IU/ml). Thereby, IFN-β pre-treatment mainly resulted in an additive anti-tumor effect in monolayer culture. Surprisingly, while IFN-β monotherapy had no effect on the colony size, it significantly enhanced the effect of gemcitabine on the colony size, suggesting a synergistic effect.
So far, there are no biomarkers for monitoring IFN activity and predicting clinical efficacy during IFN-β treatment. Booy et al. studied the correlation between the expression of the type I interferon receptor and the anti-tumor effect in a large panel of human pancreatic cancer cells. Despite the variable receptor expression among the cell lines, no significant correlation was reported regarding the maximal inhibitory effect of IFN-β.(16) Potentially, the downstream pathway of IFN can predict the response toward IFN-β treatment. In the current study, we measured the expression level of three ISGs (Mx1, IFIT1, and OAS1A), which are the functional end products of the IFN signalling pathway.(24) Therefore, their expression is induced as a result of an active IFN pathway. Consequently, expression levels of these ISGs can be assumed as a representation of activeness of the IFN pathway. At baseline, highest expression was observed in BxPC-3 and CFPAC-1, which is in line with the IFN-β sensitivity. IFN-β upregulated expression of these ISGs in all three cell lines. However, a much stronger upregulation was observed in BxPC-3 and CFPAC-1 compared to Panc-1, suggesting a less active IFN pathway in Panc-1, which might explain the lower response to IFN-β.
Expression of Mx1 was significant higher (approximately 13-fold) in the IFN-β sensitive cell lines compared to Panc-1, and strongly increased upon IFN-β treatment. The Mx1 gene encodes for the myxovirus resistant protein A (MxA) protein, which is an important antiviral factor against a wide spectrum of RNA viruses.(25) Apart from its role as a prominent antiviral protein in innate immunity, studies have indicated a role for Mx1 as a potential tumor suppressor gene. For example, deletion of Mx1 in prostate cancer is associated with a higher aggressive tendency and the expression of MxA is suppressed in a highly metastatic human prostate carcinoma cell line.(26, 27) Importantly, MxA is also employed to predict the efficacy of chemotherapy in several cancers. Knockout of Mx1 in prostate cancer cells resulted in a lower sensitivity to the chemotherapeutic agent docetaxel compared to MxA-positive cells.(28) Additionally, a study by Sistigu et al. reported a benefit of high MxA expression in patients with breast cancer receiving anthracycline-based chemotherapy.(29) Above findings indicate an important role for Mx1 in predicting the response to IFN-β treatment, as well as the potential chemosensitising effects.
So far, the effects of IFN-β, alone and combined with gemcitabine, have not been studied in animal models. By using a heterotopic subcutaneous pancreatic cancer mouse model, we are the first that confirmed the chemosensitising effect of IFN-β in vivo. Based on the response to IFN-β and the amount of IFN receptors expressed, we used the BxPC-3 cells for in vivo experiments.(16)
First, we aimed to predict therapy response before start of treatment in an ex vivo tissue slice model. The advantage of this model is the ability to evaluate multiple treatments in one tumor sample. We were able to maintain viable slices up to 4 days of culture, which is in agreement with findings of two other studies.(30, 31) Promising results were observed as the combination of IFN-β plus gemcitabine significantly reduced the proportion of Ki-67 positive cells, while apoptosis was increased in tumor tissues compared with control group.
Regarding in vivo research, the most frequently used gemcitabine concentration varies between 100 mg/kg and 125 mg/kg.(32, 33) Nevertheless, based on the previously described in vitro findings, we decided to reduce the gemcitabine concentration and used a suboptimal concentration of 40 mg/kg.
As expected, given this suboptimal treatment dose, no significant decrease of tumor volume or weight was found in mice treated with gemcitabine alone. Additionally, despite the potent anti-tumor effects in vitro, no difference was found in mice treated with IFN-β alone, however, there was a clear trend towards a smaller tumor volume. Although IFN-β concentrations were not measured in this study, it may be possible that the circulating concentration of IFN-β was not sufficient. This may be related to the relatively short half-life of IFNs in the circulation.(34) In vitro, the concentration of IFN-β required to reduce cell growth to 50% in a large series of pancreatic cancer cell lines, ranged between 70-1000 IU/ml.(16) These concentrations are not easily reached (4-10 IU/ml after four doses of 18 MIU IFN-β at 48-h intervals in serum of human healthy volunteers after s.c. administration).(34) Furthermore, the anti-tumor activities of type I IFNs can be limited by the activation of several survival pathways, such as the induction of the JAK2/STAT-3 pathway, the activation of nuclear factor kappa-beta (NF-κB) and the increased expression of the epidermal growth factor receptor (EGF-R). This could result in the stimulation of cell proliferation, malignant transformation and invasion, and the inhibition of apoptosis.(35, 36)
After 30 days of treatment, we observed a significant synergistic effect of the combined therapy of IFN-β and gemcitabine, which was reflected by the reduction of tumor volume and, additionally, by a decreased proportion of proliferating tumor cells and increased apoptosis, confirming the results observed ex vivo.
Although heterotopic subcutaneous models are often used in cancer research, it is important to evaluate the effects of IFN-β and gemcitabine in an orthotopic model as well. Especially since type I IFNs are known to induce immunoregulatory activities and interact with the tumor microenvironment.(37)
The therapeutic effectiveness of type I IFN treatment has been demonstrated in a considerable number of other malignancies, including hematologic tumors, as well as solid tumors. However, despite FDA approvals for recombinant IFN-α in a few cancers, including melanoma and renal cell carcinoma, recombinant IFN-α is not a conventional treatment for these malignancies. Long-term administration is needed to maintain therapeutic efficacy, resulting in high-grade toxicity and significant adverse side effects in patients.(38)
On the other hand, IFN-β has emerged as a safer and more potent treatment compared to IFN-α. In addition to pancreatic cancer, IFN-β induced evident anti-tumor and chemosensitising effects pre-clinically in several other cancer types, e.g. hepatocellular carcinoma and breast cancer.(39, 40) Thereby, studies report chemosensitising effects with other chemotherapeutic agents, e.g. 5-FU and cisplatin, as well, suggesting that the sensitising effect of IFN-β is not only limited to gemcitabine.(39, 41) Interesting, the intracellular uptake of these drugs are also mediated by the nucleotide transporter proteins hENT1, hCNT1, and hCNT3.(42, 43)
So far, recombinant IFN-β has not yet been approved for the treatment of any cancer type and has yet to be clinically tested in pancreatic cancer. In addition, it is recommended to study the combination of IFN-β with the new developed chemotherapeutic agents such as FOLFIRINOX and Nab-Paclitaxel as well.
While IFN therapies have been around for a while, new insights in activation of the IFN pathway have resulted in novel IFN-directed cancer treatment strategies. One example is the use of IFN based conjugates, which increase the half-life time of IFN and potentially results into higher concentrations at the tumor site.(44) In addition, the PEGylated form of IFN resulted in a higher serum concentration, requiring lower and less frequent doses compared to the conventional IFNs.(45) The PEGylated form of IFN-α has already proven to be effective in the treatment of melanoma and metastatic renal cell carcinoma patients.(46, 47) Currently, the PEGylated form IFN-β is being tested in a phase III clinical trial (ADVANCE) in patients with multiple sclerosis.(48, 49) Another novel approach is the induction of type I IFN production via activation of the STING and RIG-I pathway.(44) These approaches are currently being tested in clinical trials or are in late pre-clinical development.
Although currently no improvement has been made with immunotherapy in pancreatic cancer, IFN-β might also play a crucial role in new strategies in combination with immunotherapy. Expression of Interferon-stimulated gene 15 (ISG15) is induced by IFN-β and its pathway is highly expressed in various malignancies, including pancreatic ductal adenocarcinoma. Interestingly Burke et al. demonstrated that ISG15 pathway knockdown not only reversed the KRAS-associated phenotypes of pancreatic ductal adenocarcinoma cells, such as increased proliferation and colony formation, but also decreased tumor programmed death ligand-1 (PDL-1) expression leading to increased number of CD8+ tumor-infiltrating lymphocytes.(50)