Evangelia Skoura1,2, Konstantinos N Syrigos1, Muhammad Wasif Saif3
1Oncology Unit, Third Department of Medicine, University of Athens, Sotiria General Hospital; 2Nuclear Medicine Department, Evangelismos General Hospital. Athens, Greece.
3Tufts University School of Medicine. Boston, MA, USA
Pancreatic adenocarcinoma is an aggressive type of malignancy and remains a treatment-refractory cancer. Because of thefew treatment options, understanding of the molecular mechanisms is necessary, for new drugs be developed againstmolecular targets. Two of the novel, promising regimens against molecular targets, NVP-BEZ235 and MSK-777, wereexamined in three preclinical studies performed in human pancreatic cell lines and mouse models and presented in the 2013 ASCO Annual Meeting. Two of the studies evaluated the role of NVP-BEZ235, an oral phosphatidylinositol-3-kinase (PI3K) inhibitor, in pancreatic cancer treatment, alone and in combination with nab-paclitaxel (Abstract #e15007) or gemcitabine (Abstract #e15070). The third study presents the effectiveness of the novel cell division cycle 7 (Cdc7) kinase inhibitor, MSK- 777 (Abstract #e15059). All studies demonstrated promising results and further investigation is ongoing.
CDC7 protein, human; dactolisib; Drug Evaluation, Preclinical; Pancreatic Neoplasms
Cdc7: cell division cycle 7; MCM: minichromosome maintenance complex; NVP-BEZ235: dactolisib; PI3K: phosphatidylinositol-3-kinase
Exocrine pancreatic cancer is one of the most aggressive types of malignancy . At the time of diagnosis, most of patients present with advanced or metastatic disease . Gemcitabine has been considered as the standard treatment for advanced pancreatic cancer since 1997 . However, its efficacy remains moderate and the median overall survival times range from 5 to 8 months. Numerous studies have attempted to increase efficacy of chemotherapy by combining gemcitabine with other agents, but only the combination with erlotinib seems to induce a modest but significant increase in overall survival . In metastatic disease FOLFIRINOX (5-fluorouracil, oxaliplatin, irinotecan, leucovorin) has shown superiority over gemcitabine  while, recently in 2013, there were encouraging results in MPACT study that showed an improvement in overall survival using nabpaclitaxel plus gemcitabine versus gemcitabine alone .
Nevertheless, pancreatic adenocarcinoma remains a treatment-refractory cancer and its management remains one of the most challenging in oncology. During the last few years there have been important advances to better understand the molecular base in the development of pancreatic adenocarcinoma . The sequential acquisition of mutations in the proto-oncogene K-ras and the tumor suppressors INK4A, TP53 and DPC4/SMAD4 and their accumulation leads to a profound disturbance in cell cycle regulation and continuous growth .
Abnormal signalling by phosphatidylinositol-3- kinase (PI3K) is a prominent feature of pancreatic cancers, due to the high prevalence of abnormalities that regulate this pathway, including K-ras mutations that occur in approximately 90% of cases . It is likely that the activation of this pathway likely has an important role on the aggressive clinical features of pancreatic cancer, including resistance to chemotherapy . Genetic alterations, activating K-ras and PI3K/AKT signalling, are also known to induce the activity of mammalian target of rapamycin (mTOR) kinase in pancreatic adenocarcinoma (Figure 1) . Targeting the PI3K-AKTmTOR pathway could arrest tumor growth and induce cell death in cancers that are resistant to currently available therapies. NVP-BEZ235 (dactolisib; Pharma, Basel, Switzerland) is a synthetic low molecular mass compound belonging to the class of imidazoquinolines . It is an oral targeted anticancer agent that shows high selectivity for class I PI3K and the structurally related mTOR .
Also, studies on the molecular basis of various diseases have revealed that the deregulation of protein phosphorylation reactions by kinases is associated with many diseases, especially cancer . Cell division cycle 7 (Cdc7)-related protein kinase is a heterodimer of a kinase and its activator (Dbf4). It is essential for the activation of the minichromosome maintenance complex (MCM2-7), the helicase that unwinds the strands of DNA during replication (Figure 2) [14, 15]. Cdc7 is a target of the S-phase checkpoint, and has a role in controlling the DNA damage response. Inhibition of Cdc7 in cancer cells seems to result in slowing of S-phase and cell cycle arrest, followed by accumulation of DNA damage . Cdc7 has been shown to be overexpressed in many different tumors including the majority of solid and liquid tumors. A novel natural product small molecule inhibitor (MSK-777) has been identified, developed and shown to be highly potent in both cell based and animal models of cancer .
In the 2013 ASCO Annual Meeting were presented two abstracts concerning preclinical studies for the effectiveness of the agent NVPBEZ235 (Abstracts #e15007, #e15070) and one study for the role of MSK-777 in pancreatic cancer (Abstract #e15059).
Preclinical Study of Cytotoxicity and Predictive Markers of Response to Dual Inhibition of PI3K and mTORC1/2 Signaling by NVP-BEZ235 With or Without Paclitaxel or Nab-Paclitaxel as a New Therapeutic Strategy in Pancreatic Cancer Cell Lines (Abstract #e15007) 
Loaiza-Bonilla et al.performed a preclinical study using K-ras mutant and wild-type human pancreatic cell lines in vitro. They treated these cell lines with NVP-BEZ235 and with its combination with nab-paclitaxel. The Western blot analysis was used for S235/S236P-RPS6 and Akt (S473P-Akt, T308P-Akt) levels after the treatment. They found that although NVP-BEZ235 reduced S473P-Akt, T308P-Akt and S235/S236P-RPS6, its combination with nab-paclitaxel had a significant synergistic effect in a time- and dose-dependent fashion in comparison with the corresponding single drug treatments. These results concern only the wildtype K-ras human pancreatic cell lines (BxPC-3) but not the K-ras mutant cell lines (MIA PaCa-2 and PANC-1), where there was a paradoxical increase in proliferation.
Effect of the Combination of the Dual mTOR/PI3K Inhibitor NVP-BEZ235 with Gemcitabine on Growth Inhibition in Pancreatic Cancer Cells In Vitro and In Vivo (Abstract #e15070) 
Maute et al. examined the efficacy of NVPBEZ235 alone or in combination with gemcitabine both in vitro in human pancreatic cell lines (MiaPaCa-2, Panc-1, AsPC-1 and BxPC-3) and in vivo, in mouse models of pancreatic exocrine neoplasia (NOD SCID mice). This study showed that, in vitro, the simultaneous administration of NVPBEZ235 and gemcitabine was more effective than the single agent administration but the best results were obtained with the sequential administration of gemcitabine followed by NVP-BEZ235. In vivo, the most effective administration was the simultaneous and even better was the sequential administration of gemcitabine followed by NVP-BEZ235. Single agents (gemcitabine and NVP-BEZ235) and in the sequence NVP-BEZ235 followed by gemcitabine, resulted in a minimal reduction of tumor growth.
Cdc7 Inhibition as a Novel Approach for Pancreas Cancer Therapy (Abstract #e15059) 
Frattini et al. examined the effectiveness of MSK- 777 in pancreatic cancer. They used three cell lines, Capan-1, BxPC3, and PANC-1 and cytotoxicity of the regimen was estimated. MSK-777, control (dimethyl sulfoxide; DMSO), or hydroxyurea was administrated. After the first 24 hours in BxPC3 cells there was a dramatic effect with reduced cell viability to less than 20%. Also, the study demonstrated that in Capan-1 and PANC-1 cells, the administration of MSK-777 resulted in cell cycle arrest at G1/S by 48 hours while in the BxPC3 cells, there was a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Concerning the phosphorylation of MCM-2, in BxPC3 cells it disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication, while in Capan-1 and PANC-1 cells there were lower levels of phosphorylated MCM-2 by 48 hours
Pancreatic cancer represents the fifth leading cause of cancer death in the Western population with a five-year survival rate under 5% . Because of the few treatment options, understanding of the molecular mechanisms is prerequisite to identify potential molecular targets for drug therapy . The identification of novel molecular targets is critical in the development of new and efficient cancer therapies . Two promising regimen, NVP-BEZ235 and MSK-777 were presented in ASCO 2013 Annual Meeting
Concerning the role of NVP-BEZ235 in treatment of pancreatic adenocarcinoma, only a few preclinical studies have been published. The first of them, performed in 2009, showed that this agent might have anticancer activity but the authors supported the testing of combination of NVPBEZ235 with other anticancer agents . Also, more recent studies using NVP-BEZ235, demonstrated that it is unlikely for the complete target inhibition to be sustained over long periods, as PI3K signalling is critical in the maintenance of normal tissues. So combination of this agent with other anticancer agents was studied [10, 20]. So, in 2012, two preclinical studies showed that NVP-BEZ235 alone could effectively induced apoptosis and reduced tumor growth, but the effectiveness was further enhanced by combination of gemcitabine and endothelial monocyte activating polypeptide II (EMAP)  or by combination with the panhistone deacetylase inhibitor panobinostat (PS) . In agreement with these results, Loaiza-Bonilla et al. showed that NVP-BEZ235 in combination with nab-paclitaxel had a significant synergistic effect compared to monotherapy . This effectiveness was demonstrated only in cancer cell lines with mutant K-ras and not in wild type. The authors resumed that K-ras may be studied as a potential predictive biomarker. The second study concerning the effectiveness of NVP-BEZ235 was performed from Maute et al.  and they used the combination of NVP-BEZ235 of gemcitabine. They used not only in vitro analysis (cell lines) but also in vivo experiments in NOD SCID mice. Although, the combination of the two regimens seemed to work significantly better than the single agents, it is interesting that the most effective was the sequential administration of gemcitabine followed by NVP-BEZ235, in both in vitro and in vivo analysis. The above promising results may offer the opportunity of a more effective treatment of pancreatic cancer.
Cdc7 is overexpressed in most cancers, including many solid tumors and acute leukemias. For the Cdc7 inhibitor MSK-777, examined in the study by Frattini et al. , there are not published data until today, as it is a novel regimen identified in the researchers’ laboratory, while their first efforts focused in treatment of leukemia . Studies have shown that Cdc7 inhibition in cancer cells leads to p53-independent apoptotic cell death, as it produces cells bearing incompletely and/or abnormally replicated DNA, which results in cell death . It is interesting that Cdc7 depletion does not elicit apoptotic cell death in normal human diploid cells, as it seems that there is a cell-cycle check-point mechanism . The preclinical data showed that Cdc7 is a novel and promising target for tumor cell killing but ongoing trials will reveal whether inhibition of this kinase represents a successful strategy that could bring benefits to cancer patients in terms of superior activity and better tolerability compared with the currently approved agents [13, 21].
In conclusion, although during the last few years there have been important advances in the understanding of the molecular events responsible for the development of pancreatic cancer, currently specific mechanisms of treatment resistance remain poorly understood and new effective systemic drugs need to be developed and probed. The results of the above presented studies in 2013 ASCO Annual Meeting, show that NVP-BEZ235 and MSK- 777 represent novel and promising treatment, although they have to be tested in further studies.
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