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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.

Pancreatic ductal adenocarcinoma is one of the lethal cancers with extensive local tumour invasion, metastasis, early systemic dissemination and poorest prognosis. Thus, understanding the mechanisms regulating invasion/metastasis and epithelial–mesenchymal transition (EMT), is the key for developing effective therapeutic strategies for pancreatic cancer (PaCa). Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase that we found to be highly up-regulated in PaCa cells. However, its role in PaCa invasion/progression remains unknown. Here, we investigated the role of eEF-2K in cellular invasion, and we found that down-regulation of eEF-2K, by siRNA or rottlerin, displays impairment of PaCa cells invasion/migration, with significant decreases in the expression of tissue transglutaminase (TG2), the multifunctional enzyme implicated in regulation of cell attachment, motility and survival. These events were associated with reductions in β1 integrin/uPAR/MMP-2 expressions as well as decrease in Src activity. Furthermore, inhibition of eEF-2K/TG2 axis suppresses the EMT, as demonstrated by the modulation of the zinc finger transcription factors, ZEB1/Snail, and the tight junction proteins, claudins. Importantly, while eEF-2K silencing recapitulates the rottlerin-induced inhibition of invasion and correlated events, eEF-2K overexpression, by lentivirus-based expression system, suppresses such rottlerin effects and potentiates PaCa cells invasion/migration capability. Collectively, our results show, for the first time, that eEF-2K is involved in regulation of the invasive phenotype of PaCa cells through promoting a new signalling pathway, which is mediated by TG2/β1 integrin/Src/uPAR/MMP-2, and the induction of EMT biomarkers which enhance cancer cell motility and metastatic potential. Thus, eEF-2K could represent a novel potential therapeutic target in pancreatic cancer.

Advances in cancer therapeutics, namely more effective and less toxic treatments, will occur with targeting strategies that enhance the tumor biodistribution and thwart normal tissue exposure of the drug. This review focuses on cancer drug targeting approaches that exploit the expression of the cell-surface proteoglycan family, CD44, on the tumor cell surface followed by some form of ligand binding and induced CD44 internalization and intracellular drug release: in effect using this as a 'Trojan Horse' to more selectively access tumor cells.This review defines the origins of evidence for a linkage between CD44 expression and malignancy, and invokes contemporary views of the importance of putative CD44(+) cancer stem cells in disease resistance. Although the primary emphasis is on the most advanced and developed paths, those that have either made it to the clinic or are well-poised to get there, a wide scope of additional approaches at various preclinical stages is also briefly reviewed.The future should see development of drug targeting approaches that exploit CD44 expression on CSCs/TICs, including applications to cytotoxic agents currently in the clinic.

Pancreatic cancer (PaCa) is one of the most aggressive, apoptosis-resistant and currently incurable cancers with a poor survival rate. Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase, whose role in PaCa survival is not yet known. Here, we show that eEF-2K is overexpressed in PaCa cells and its down-regulation induces apoptotic cell death. Rottlerin (ROT), a polyphenolic compound initially identified as a PKC-δ inhibitor, induces apoptosis and autophagy in a variety of cancer cells including PaCa cells. We demonstrated that ROT induces intrinsic apoptosis, with dissipation of mitochondrial membrane potential (ΔΨm), and stimulates extrinsic apoptosis with concomitant induction of TNF-related apoptosis inducing ligand (TRAIL) receptors, DR4 and DR5, with caspase-8 activation, in PANC-1 and MIAPaCa-2 cells. Notably, while none of these effects were dependent on PKC-δ inhibition, ROT down-regulates eEF-2K at mRNA level, and induce eEF-2K protein degradation through ubiquitin–proteasome pathway. Down-regulation of eEF-2K recapitulates the events observed after ROT treatment, while its over-expression suppressed the ROT-induced apoptosis. Furthermore, eEF-2K regulates the expression of tissue transglutaminase (TG2), an enzyme previously implicated in proliferation, drug resistance and survival of cancer cells. Inhibition of eEF-2K/TG2 axis leads to caspase-independent apoptosis which is associated with induction of apoptosis-inducing factor (AIF). Collectively, these results indicate, for the first time, that the down-regulation of eEF-2K leads to induction of intrinsic, extrinsic as well as AIF-dependent apoptosis in PaCa cells, suggesting that eEF-2K may represent an attractive therapeutic target for the future anticancer agents in PaCa.

The androgen receptor (AR) and the phosphoinositide 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin (mTOR) signaling are two of the major proliferative pathways in a number of tissues and are the main therapeutic targets in various disorders, including prostate cancer (PCa). Previous work has shown that there is reciprocal feedback regulation of PI3K and AR signaling in PCa, suggesting that cotargeting both pathways may enhance therapeutic efficacy. Here we show that proteins encoded by two androgen-regulated genes, kallikrein related peptidase 4 (KLK4) and promyelocytic leukemia zinc finger (PLZF), integrate optimal functioning of AR and mTOR signaling in PCa cells. KLK4 interacts with PLZF and decreases its stability. PLZF in turn interacts with AR and inhibits its function as a transcription factor. PLZF also activates expression of regulated in development and DNA damage responses 1, an inhibitor of mTORC1. Thus, a unique molecular switch is generated that regulates both AR and PI3K signaling. Consistently, KLK4 knockdown results in a significant decline in PCa cell proliferation in vitro and in vivo, decreases anchorage-independent growth, induces apoptosis, and dramatically sensitizes PCa cells to apoptosis-inducing agents. Furthermore, in vivo nanoliposomal KLK4 siRNA delivery in mice bearing PCa tumors results in profound remission. These results demonstrate that the activities of AR and mTOR pathways are maintained by KLK4, which may thus be a viable target for therapy.

Translation initiation and activity of eukaryotic initiation factor-alpha (eIF2α), the rate-limiting step of translation initiation, is often overactivated in malignant cells. Here, we investigated the regulation and role of eIF2α in acute promyelocytic (APL) and acute myeloid leukemia (AML) cells in response to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), the front-line therapies in APL. ATRA and ATO induce Ser-51 phosphorylation (inactivation) of eIF2α, through the induction of protein kinase C delta (PKCδ) and PKR, but not other eIF2α kinases, such as GCN2 and PERK in APL (NB4) and AML cells (HL60, U937, and THP-1). Inhibition of eIF2α reduced the expression of cellular proteins that are involved in apoptosis (DAP5/p97), cell cycle (p21Waf1/Cip1), differentiation (TG2) and induced those regulating proliferation (c-myc) and survival (p70S6K). PI3K/Akt/mTOR pathway is involved in regulation of eIF2α through PKCδ/PKR axis. PKCδ and p-eIF2α protein expression levels revealed a significant association between the reduced levels of PKCδ (P=0.0378) and peIF2 (P=0.0041) and relapses in AML patients (n=47). In conclusion, our study provides the first evidence that PKCδ regulates/inhibits eIF2α through induction of PKR in AML cells and reveals a novel signaling mechanism regulating translation initiation.

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

Summary: Therapeutic upregulation of macroautophagy in cancer cells provides an alternative mechanism for cell death. Prolactin (PRL) and its receptor (PRLR) are considered attractive therapeutic targets because of their roles as growth factors in tumor growth and progression. We utilized G129R, an antagonist peptide of PRL, to block activity of the tumoral PRL/PRLR axis, which resulted in inhibition of tumor growth in orthotopic models of human ovarian cancer. Prolonged treatment with G129R induced the accumulation of redundant autolysosomes in 3D cancer spheroids, leading to a type II programmed cell death. This inducible autophagy was a noncanonical beclin-1-independent pathway and was sustained by an astrocytic phosphoprotein (PEA-15) and protein kinase C zeta interactome. Lower levels of tumoral PRL/PRLR in clinical samples were associated with longer patient survival. Our findings provide an understanding of the mechanisms of tumor growth inhibition through targeting PRL/PRLR and may have clinical implications. : Pharmacological manipulation of autophagy represents a new therapeutic opportunity for cancer. Wen et al. show that blockade of the tumoral PRL/PRLR axis with an antagonist, G129R, induces prolonged autophagy. This inducible autophagy is sustained by the PEA-15 and PKC zeta interactome and leads to type II programmed cell death. There was an inverse correlation between tumoral PRL/PRLR expression and survival of ovarian cancer patients. This study reveals a previously unrecognized mechanism related to targeting the tumoral PRL/PRLR pathway.