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SummaryWhile recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo’s effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin.

Breast cancer is the second leading cause of tumor related death in women in Western countries. It has been estimated in recent reports that this year about 207.000 women will be diagnosed with breast cancer and about 40.000 will die of it in the US. (Jemal et al., 2010). While 5 year survival in early stages is about 90 %, it is as low as 15 % in metastatic stage. Despite the fact that there are many agents to treat the breast cancer, most of the tumors ultimately become unresponsive to these systemic therapeutics (Alvarez et al., 2010). Therefore new therapeutic strategies either alone or in combination with conventional therapies are required to improve the survival rates of breast cancer patients.

Ovarian cancer (OC) and Pancreatic ductal adenocarcinoma (PDAC) are the most aggressive and deadliest cancers and is currently incurable disease with the poorest prognosis and survival rates (4% and 30% 5-year survival rates, respectively). The poor prognosis is attributed to the extensive local tumor invasion and the resistance to existing cancer therapeutics, which are the major characteristics of both cancers and the impediment to effective cure this lethal diseases. Although both cancers have a well-defined spectrum of highly recurrent oncogenic lesions, including p53, c-myc, K-ras, CDKN2A /p16, TP53 and SMAD4/DPC4 effective therapies have not yet been developed. Therefore, novel molecular targets and therapeutics strategies are urgently needed. Recently, we discovered that Eukaryotic elongation factor-2 kinase (EF2K), an atypical kinase, is dramatically up-regulated in cells and promotes cell survival and proliferation, its inhibition significantly reduces cell growth and colony formation. However, the role that EF2K in these cancers and pathways regulating survival, invasion/metastasis and resistance remains is largely unknown. Here, we show that specific inhibition of EF2K by siRNA-mediated knock markedly inhibits OC (HeyA8 and SKOV3ip1) and PDAC (PANC1 and MiaPaca-2) invasion in in vitro matrigel assay. Furthermore, inhibit EF2K expression, also significantly reduced tissue transglutaminase (TG2), a multifunctional enzyme implicated in regulation of cell survival, migration and invasion and beta-integrin. We found that eEF-2K regulates TG2, at the transcription level, as evidenced by eEF-2K knockdown-mediated marked down-regulation of TG2 protein and mRNA levels by Western blot and RT-PCR, respectively. Lack of function and overexpression (by Lenti-based system) demonstrated that EF2K regulates TG2,Beta-1 integrin, uPAR MMP-2, p-SRC expression. In addition, we found down-regulation of EF2K or TG2 by siRNA suppresses the downstream key cellular pathways supporting invasion, including epithelial-mesenchymal transition (EMT) through the modulation of the zinc finger transcription factors, belonging to ZEB1 family, and the tight junction proteins, claudins. Furthermore, we found that therapeutic inhibition of EF2K by systemically administered (i.v) nano liposomal EF2K siRNA inhibited growth SKOV3and intraperitoneal tumor nodules (intraperitoneal metastatic model) Panc1 tumor xenografts in nude mice (p Citation Format: Ahmed Ashour, Alper Erdogan, Sultan Neslihan Alpay, Nermin Kahraman, Erkan Yuka, Gabriel Lopez-Berestein, Bulent Ozpolat. EF2-kinase (EF2K): A novel molecular target in ovarian and pancreatic cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2642. doi:10.1158/1538-7445.AM2015-2642

Pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of pancreatic cancer (PaCa) cases and is currently uncurable with poor prognosis/survival rates (∼4% 5-year survival). The extensive local tumor invasion, early metastasis/systemic dissemination and resistance to existing cancer therapies are the major characteristics of PaCa and the impediment to effective cure of this disease. Although PaCa has a well-defined spectrum of highly oncogenic lesions (e.g, mutations in K-RAS, p53, p16ink4a, and SMAD4/DPC4), effective therapies have not yet been developed. Therefore, novel molecular targets-based therapeutic strategies are urgently needed. Recently, we discovered that eukaryotic elongation factor-2 kinase (eEF-2K) is dramatically up-regulated in different PaCa cells, promoting their survival, and that targeted-silencing of eEF-2K led to significant cell death (Ashour et al, 2013). However, the role of eEF-2K in the pathways regulating invasion/metastasis and drug-resistance remains largely unknown. Here, we show that siRNA-mediated knockdown of eEF-2K markedly inhibits the invasion of PANC-1and MIAPaCa-2 cells. Furthermore, rottlerin, which we found to down-regulate eEF-2K expression, also significantly reduced the invasion of both cell lines. We later investigated the involvement of tissue transglutaminase (TG2), a multifunctional enzyme implicated in regulation of cell survival, migration and invasion. We found that eEF-2K regulates TG2 at the transcription level, as evidenced by eEF-2K knockdown-mediated down-regulation of TG2 protein and mRNA levels by Western blot and RT-PCR, respectively. Importantly, siRNA-mediated knock-down of eEF-2K or TG2 separately, suppresses the downstream key cellular pathways supporting PaCa invasion, and recapitulates rottlerin-induced invasion inhibition and correlated events. We also found that inhibition of eEF-2K/TG2 axis suppresses the epithelial-mesenchymal transition (EMT) through the modulation of ZEB1 zinc finger transcription factors, and tight junction proteins, claudins. Moreover, eEF-2K overexpression, by lentivirus-based expression system, led to increased TG2 expression, with concomitant induction in the invasion capability of PaCa cells. eEF-2K overexpression also rescued the cells from rottlerin-induced suppression of invasion and invasion-supporting machineries, suggesting that eEF-2K is a prominent target of rottlerin, and is a central regulator of PaCa cell invasion. We are currently treating PANC-1 tumor xenografts in nude mice with eEF-2K siRNA-nanoparticles to demonstrate the role of eEF-2K in invasion and tumor progression, in order to establish eEF-2K as a potential novel therapeutic target in PaCa. Collectively, our data suggest, for the first time, the pivotal role of eEF-2K in PaCa invasion/progression and possibly tumor growth. Citation Format: Ahmed A. Ashour, Sultan N. Alpay, Nilgun Gurbuz, Abdel-Aziz H. Abdel-Aziz, Ahmed M. Mansour, Bulent Ozpolat. Elongation factor-2 kinase (eEF-2K) promotes cell invasion and epithelial mesenchymal transition through regulation of TG2-mediated signaling in human pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2014-4054

Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa) patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT) contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA), induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT), concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D- mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification, and valuable new therapeutic targets for managing pancreatic cancer.

Pancreatic cancer (PaCa) is one of the most aggressive cancers and currently incurable with less than 2% five-years survival rate. Among the ever-increasing list of naturally occurring anticancer agents, a Kmala Tree-derived anticancer compound, Rottlerin (ROT), a PKC-delta inhibitor, appears to have anti-proliferative activity due to its effects on several pathways and cell machineries involved in cell survival, apoptosis, autophagy and invasion. Recent studies suggest that ROT is not a specific PKC delta inhibitor and mediates its effects through other mechanisms. We have previously reported that inhibition of eukaryotic elongation factor-2 kinase (eEF-2K), one of the major kinases appears to be activated in rapidly proliferating malignant cells, leads to down-regulation of signaling pathways affecting growth, survival and chemotherapeutic resistance (Tekedereli et al, 2012). Thus, we hypothesized that ROT targets eEF-2K, and inhibition of eEF-2K signaling is responsible of mediating its effects in PaCa cells. We found that ROT treatment (4-10 μM) inhibits the expression of eEF-2K that was associated with apoptosis induction in PaCa cells in a dose and time-dependent manner as detected by Western blot analysis. We also found that ROT treatment inhibits PaCa cell proliferation and mitochondrial activity and modulates G1/S phase progression in these cells. Treating PaCa cells with ROT markedly induced expression of active caspase-9, caspase-3 and cleaved PARP. To demonstrate a direct link between eEF-2K inhibition and ROT-induced effects, we knocked-down eEF-2K by a specific siRNA. Such knockdown was associated with inhibition of cell growth, resulted in modulation of caspase-related events with induction of similar degree of apoptosis, increased the percentage of Annexin V positive apoptotic cells and resulted in induction of cellular shrinkage and blebbing. Moreover, we found that eEF-2K down-regulation is involved in ROT-induced stimulation of extrinsic apoptotic pathway in PaCa cells with concomitant induction of TNF related apoptosis inducing ligand (TRAIL) receptor DR4 and caspase-8 cleavage. In addition, our findings suggest that eEF-2K contributes to the regulation of G1/S phase progression, as in vitro down-modulation eEF-2K resulted in an increase in the expression of p27Kip1 cyclin-dependent kinase inhibitor (CDKI). Collectively, the results of our study demonstrate, for the first time, that down-regulation of eEF-2K contributes to rottlerin-induced effects including, growth inhibition and apoptosis in PaCa cells and clearly demonstrate novel mechanisms of ROT-induced potent antitumor effects. These findings show that the multi-targeted kinase inhibitor, ROT, represents a promising novel agent for PaCa treatment and eEF-2K could represent an attractive target for the future anticancer agents. Citation Format: Ahmed A. Ashour, Abdel-Aziz H. Abdel-Aziz, Ahmed M. Mansour, Sultan N. Alpay, Kevin Dalby, Bulent Ozpolat. Inhibition of eukaryotic elongation factor-2 kinase mediates Rottlerin induced effects in apoptosis and cell proliferation inhibition in human pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 848. doi:10.1158/1538-7445.AM2013-848

Eukaryotic elongation factor 2 kinase (eEF-2K), through its phosphorylation of elongation factor 2 (eEF2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. The purpose of this study was to examine the therapeutic potential of targeting eEF-2K in breast cancer tumors. Through the systemic administration of liposomal eEF-2K siRNA (twice a week, i.v. 150 µg/kg), the expression of eEF-2K was down-regulated in vivo in an orthotopic xenograft mouse model of a highly aggressive triple negative MDA-MB-231 tumor. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth, the induction of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin. eEF-2K down-modulation in vitro resulted in a decrease in the expression of c-Myc and cyclin D1 with a concomitant increase in the expression of p27(Kip1). A decrease in the basal activity of c-Src (phospho-Tyr-416), focal adhesion kinase (phospho-Tyr-397), and Akt (phospho-Ser-473) was also detected following eEF-2K down-regulation in MDA-MB-231 cells, as determined by Western blotting. Where tested, similar results were seen in ER-positive MCF-7 cells. These effects were also accompanied by a decrease in the observed invasive phenotype of the MDA-MB-231 cells. These data support the notion that the disruption of eEF-2K expression in breast cancer cells results in the down-regulation of signaling pathways affecting growth, survival and resistance and has potential as a therapeutic approach for the treatment of breast cancer.

Breast cancer is the second most common cause of cancer death in women in the United States, 40,000 dying of this disease each year. One out of eight women will be diagnosed with breast cancer in their lifetime. There are several treatments for breast cancer, such as surgery, radiation therapy, chemotherapy, and hormone therapy, but still 5 year survival is less than 40% in advance cases. Due to the high incidence and mortality associated with this cancer better understanding of the biology of the disease and development of new treatments are urgently needed. The actions of local physiological mediators in the context of breast cancer are poorly understood. Serotonin (5HT, 5-hydroxytryptamine) is a critical local regulator of epithelial homeostasis in the breast and other organs. Serotonin is more commonly known as a monoamine neurotransmitter and a mediator of depression if the levels drop in the brain. Serotonin has been implicated in epithelial homeostasis of the breast, lung, pancreas, liver, and prostate. Deregulation of serotonin signaling in these epithelia may be associated with various pathologies, including cancer. Previous and our studies suggested serotonin can induce proliferation of some cancer cell lines. On the other hand serotonin can induce apoptosis or induce cell proliferation depending on the cancer type. Serotonin is believed to function as a growth factor working as an autocrin manner. Also tryptophan hydroxylase 1 (TPH1), rate-limiting step in 5-HT synthesis, has been shown to be overexpressed in breast cancer cell lines and biopsy samples from patients. The effect of serotonin signaling and its role in survival of breast cancer cells has not been well understood. We tried to address the questions that what is the role of serotonin signaling in breast cancer survival and if serotonin inhibits apoptosis or autophagy? We found that serotonin slightly induced cell proliferation in estrogen receptor positive MCF-7 breast cancer cells. Because autophagy is implicated as a survival pathway in some cancers we also examined if serotonin induces autophagy in MCF7 ER+ breast cancer cells. We found that serotonin induces autophagy that is detected Acridine orange staining and FACS analysis and LC-3 II induction, a hallmark of autophagy, by western blot analysis in MCF7 cells after serotonin treatment for 4 hours. We found that serotonin (10 uM) induces expression of cyclinD1, NF-kB and mTOR and leads induction of ERK and AMPK pathway as indicated by increased phosphorylation of ERK1/2 and AMPK (Thr 172), both of which are known to mediate autophagy. We are currently testing if induction ERK and AMPk signaling is involved in induction autophagy in these cells using specific inhibitors of MEK. In conclusion our studies showed for the first that induction of serotonin signaling induces autophagy in ER+ breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3787. doi:10.1158/1538-7445.AM2011-3787