Enhancing sorafenib-mediated sensitization to gemcitabine in experimental pancreatic cancer through EMAP II

Pancreatic ductal adenocarcinoma (PDAC) remains a deadly human cancer with very poor prognosis and a 5-year survival of less than 5% [1]. This is primarily related to its late clinical presentation, early and aggressive local or metastatic progression and high resistance to conventional chemotherapy and radiation treatments. Gemcitabine (Gem), a cytotoxic nucleoside analog, is the most widely used single agent chemotherapeutic treatment for locally advanced and metastatic PDAC [2]. The efficacy of gemcitabine remains modest with a median survival of approximately 6 months and one-year survival of less than 20% [2‐4]. Currently several clinical studies are underway to explore combination treatment benefits of gemcitabine with other cytotoxic, antiangiogenic or targeted agents for novel and more effective therapeutic strategies for PDAC. In addition, FOLFIRINOX is a combination cytotoxic regimen that has shown a somewhat greater efficacy but also greater toxicity potential compared to gemcitabine [5].

The K-ras oncogene is mutated in up to 90% of PDAC [6‐8], leading to constitutive activation of the Ras/Raf/MEK/ERK signal transduction pathway and suggesting that this pathway could represent an important target for PDAC therapy. Sorafenib (So, Nexavar, BAY 43-9006) is a novel, potent, orally available multikinase inhibitor targeting Raf serine/threonine kinases as well as different receptor tyrosine kinases including vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor receptor (PDGFR), c-Kit, FLT-3 and RET [9, 10]. In preclinical studies sorafenib has shown significant antitumor responses in several tumor types including renal cell carcinoma, pancreatic cancer, colon cancer, breast cancer and melanoma based in part on its inhibitory effect on the Ras/Raf/MEK/ERK and angiogenesis pathways [9‐11]. Sorafenib is approved for the clinical treatment of hepatocellular carcinoma and renal cell carcinoma [12]. A phase I trial of sorafenib plus gemcitabine in advanced PDAC showed that this combination was well tolerated and that 57% patients experienced stable disease [13]. More recently, a phase II trial of sorafenib plus gemcitabine showed no significant clinical activity in advanced PDAC [14]. These results support an evaluation of the addition of other antitumor agents to sorafenib plus gemcitabine for targeting multiple pathways that partake in PDAC progression.

Activated angiogenesis mechanisms are essential for the progression of primary and metastatic solid tumors including PDAC. Antiangiogenic agents including bevacizumab, an antibody against vascular endothelial growth factor (VEGF) [15, 16], the matrix metalloproteinase inhibitor marimastat [17], the cyclooxygenase-2 inhibitor celecoxib [18] and various other TKIs [19] have been tested clinically in PDAC with limited survival benefit [20]. Endothelial monocyte activating polypeptide II (EMAP, E) is a proinflammatory cytokine with antiangiogenic and antiendothelial activities. Although EMAP has no effect on in vitro AsPC-1 PDAC cell line proliferation or apoptosis [21, 22], it has potent effects on endothelial cells (ECs) such as inhibition of proliferation, migration and vascularization as well as induction of apoptosis [23, 24]. EMAP has been shown to suppress primary and metastatic tumor growth [23, 25, 26] that could be related to its ability to bind VEGF receptors and α5β1 integrin, leading to interference in fibronectin- and VEGF signaling [27, 28]. EMAP has recently been shown to improve gemcitabine and docetaxel response in experimental PDAC [21, 29, 30]. In the present study, we tested the hypothesis that combination treatment of EMAP with sorafenib and gemcitabine can enhance antitumor effects by blocking multiple critical pathways leading to progression of PDAC, to define an option for future PDAC clinical applications.

PDAC shows limited susceptibility to almost all classes of cytotoxic drugs. Several molecular genetic abnormalities in PDAC are being encountered with a high frequency, including activating K-ras mutation, loss of p16, p53 and DPC4 (deleted in pancreatic cancer, locus 4) function, and over-expression of multiple receptor tyrosine kinases [36, 37]. Tumor heterogeneity resulting from the diverse molecular abnormalities acquired during malignant transformation creates a rationale to evaluate multi-targeted therapeutic strategies against many human malignancies including PDAC. Sorafenib is a novel, potent, small molecular mass inhibitor with combined anticancer activities through the inhibition of tumor cell proliferation and tumor angiogenesis. Combining conventional cytotoxic drugs, such as gemcitabine, with targeted agents that specifically interfere with key operational pathways responsible for PDAC progression, such as sorafenib, is gaining more traction in the efforts to identify more effective combination treatments for PDAC.

In PDAC progression, angiogenesis plays a critical role that is highly dependent on the complex interaction among tumor cells, ECs, immune cells, fibroblasts and other stromal components, all contributing to the well-characterized extensively desmoplastic and hypoxic local tumor microenvironment of pancreatic cancer. Specifically for this reason, antiendothelial and antiangiogenic agents may be beneficial in combination therapy approaches for PDAC treatment. In the present study we evaluated the antitumor activity of sorafenib, and the enhancement of gemcitabine response by addition of sorafenib and the antiangiogenic agent EMAP in experimental pancreatic cancer. We demonstrate that in PDAC cells sorafenib treatment effectively blocked phosphorylation of MEK (Ser221), ERK1/2 (Thr202/Tyr204) and downstream target proteins phospho-p70 S6K (Thr389) and phospho-4E-BP1 (Thr37/46) in most of the cell lines tested except BxPC-3, where upstream MEK and ERK phosphorylation was inhibited but not the downstream signaling proteins p70S6K or 4-EBP-1. These findings suggest that sorafenib may cause some specific effects that result in blockage of Ras/Raf/MEK/ERK signaling and interfere with pancreatic cancer cell proliferation, differentiation and survival. Sorafenib treatment decreased cell proliferation and induced apoptosis in ECs and fibroblasts indicating that the in vivo antitumor effects of sorafenib may be due to its direct cytotoxic effects on various tumor cellular components, in addition to its antiangiogenic properties.

Previous studies have shown marked heterogeneity in gemcitabine and other chemotherapeutic agent response towards PADC cells [38‐40]. We also observed a heterogeneous response of sorafenib and gemcitabine in inhibiting cell proliferation of four PDAC lines tested. Both agents caused inhibition of cell proliferation to different extents and the addition of sorafenib improved gemcitabine effects. Effects of combinations of EMAP with sorafenib and gemcitabine were evaluated in ECs and fibroblast cells, and a significant additive effect on inhibition of cell proliferation was observed compared with single or dual agent treatment. A gemcitabine plus sorafenib combination was found to be effective in preclinical and phase I trials of PDAC, lending support to the importance of combining cytotoxic drugs with agents inhibiting Ras/Raf/MEK/ERK pathways and angiogenesis [9‐11, 13]. However, a phase II trial showed no meaningful effect of the gemcitabine plus sorafenib combination in advanced PDAC patients [14]. The very small number of 17 patients and 94% of patients carrying metastatic disease were the contributing factors in the negative phase II clinical trial results [14]. These results also indicate the importance of targeting other relevant pathways that contribute in the progression of PDAC. Currently, two phase II trials are evaluating the combination treatment benefits of gemcitabine, sorafenib and the EGFR inhibitor erlotinib in advanced PDAC. The anti-vascular endothelial growth factor agent bevacizumab, the first FDA-approved angiogenesis inhibitor, showed promising phase II data in combination with gemcitabine in PDAC patients but failed to demonstrate any survival benefit in phase III trials [41]. Since sorafenib inhibits the raf kinase and VEGF pathways, we assumed that the addition of EMAP, an inhibitor of VEGF and integrin-fibronectin pathways [25, 27], to gemcitabine and sorafenib would potentially improve in vivo outcome of clinical PDAC. This assumption was based on the effective in vitro combination data with EMAP in previous studies showing EMAP enhancing antitumor effects of gemcitabine paired with bevacizumab [21] or with the mTOR and AKT inhibitor NVP-BEZ235 [40].

Activating K-ras mutations are highly prevalent and have been shown to be important in the initiation and progression of pancreatic cancer. Farnesyltransferase inhibitors that can block K-ras activation have been tested clinically, but the results showed insufficient antitumor activity perhaps indicating the importance of multi-targeted strategies against PDAC that can extend beyond the inhibition of a single upstream mediator within a frequently activated signaling pathway [42]. Later studies focused on therapeutic targeting of the Ras/Raf/MEK/ERK network in combination with other important molecular targets by multikinase inhibitors such as sorafenib that has been shown to generate some antitumor activity as single agent in a pancreatic cancer cells [43]. Our results not only corroborate with these findings, but also demonstrate the impact of sorafenib and its combinations with gemcitabine on several other, potentially relevant cell types and on experimental PDAC survival. In addition, we tested combination treatment benefits of sorafenib with gemcitabine and EMAP, based on previous studies in our lab that showed EMAP-derived improvements of gemcitabine effects in vivo [29, 31]. The observed advantages of combining these agents can be interpreted as supportive of a rationale to a multi-agent clinical approach to PDAC that includes a multikinase inhibitor, a targeted multi-pathway blocker such as sorafenib, and an antiendothelial or antiangiogenic agent. Although optimal combination conditions and exact mechanisms are still not clear, these findings may provide a solid foundation for future evaluation of combination benefits of agents displaying these known effects.

Based on the limited efficacy of sorafenib in a therapeutic approach confined to 2 weeks, prolonged or intermittent dosing could be considered as an option for achieving progression-free benefits more likely. While we have not tested this approach in our experiments to date, there is concern over the true ability to obtain superior antitumor effects in the long term. Other than the commonly known side effects that could prevent this from being a clinically feasible strategy, persistent long term use of sorafenib might also lead to the development of resistant tumor cells with a more aggressive phenotype due to some epithelial-to-mesenchymal transition (EMT) at the time of tumor recurrence [44]. Therefore an altered/decreased dose of a multikinase inhibitor such as sorafenib in combination with a chemotherapeutic and antiangiogenic/targeted agent may provide a better therapeutic option.

In summary, our present study demonstrates that the multikinase inhibitor sorafenib, either alone or in combination with gemcitabine and EMAP, induced strong antiproliferative and proapoptotic effects in vitro. While the in vivo effects of sorafenib were limited, the addition of EMAP enhanced the combination treatment of sorafenib and gemcitabine in improving animal survival. This provides evidence that targeting multiple mechanisms of pancreatic cancer progression can be a promising therapeutic approach for PDAC treatment.