Activity In Pancreatic Cancer Cells Research Articles

Genistein induced anticancer effects on pancreatic cancer cell lines involves mitochondrial apoptosis, G0/G1cell cycle arrest and regulation of STAT3 signalling pathway

BackgroundGenistein is a natural flavonoid that has been reported to exhibit anticancer effects against different types of cancers which include, but are not limited to, breast and oral squamous cell carcinoma. PurposeThe present study was designed to evaluate the anticancer effects of the natural flavonoid genistein against pancreatic cancer cell lines and to explore the underlying mechanism. MethodsAntiproliferative activity was investigated by MTT assay. Apoptosis was detected by DAPI and annexin V/PI staining. DNA damage was assessed by comet assay. Reactive oxygen species (ROS) and reduction of mitochondrial membrane potential (MMP) were determined by flow cytometry. Cell migration was examined by wound healing assay. Protien expressions were determined by western blotting. ResultsAntiproliferative assay revealed that genistein reduced the cell viability of pancreatic cancer cells in a dose dependent manner with an IC50 of 20 and 25 µM against Mia-PaCa2 and PANC-1 cancer cell lines respectively. However, its antiproliferative effects were less pronounced against non-cancerous pancreatic ductal epithelial cell line (H6C7) as evident from the IC50 of 120 µM. Genistein induced significant morphological changes in pancreatic cancer cells and triggered cell cycle arrest in G0/G1 phase. DAPI staining and flow cytometric analysis revealed that genistein induced apoptosis in a dose dependent manner through generation of substantial amounts of ROS and reduction of MMP. However, treatment of the pancreatic cancer with genistein and ascorbic acid could abrogate the effects of genistein on cell viability. Protien expression analysis revealed that genistein upregulated cytosolic cytochrome c, Bax, cleaved Caspase-3 and cleaved caspase-9 expressions with concomitant downregulation of Bcl-2 expression. Moreover, genistein inhibited the phosphorylation of signal transducer and activator of transcription STAT3 proteins and downregulated the expression of survivin, cyclin D1 and ALDH1A1 in Mia-PaCa2 cells in a dose dependent manner. Interestingly, genistein could inhibit the cell migration potential of the Mia-PaCa2 cells which was further associated with the downregulation of metalloproteinases (MPP-2 and MPP-9). ConclusionTaken together, we propose that genistein exerts anticancer activity in pancreatic cancer cells through induction of ROS mediated mitochondrial apoptosis, cell cycle arrest and regulation of STAT3 and may therefore prove beneficial in the management of pancreatic cancers cancer.

Novel histone deacetylase inhibitor AR-42 exhibits antitumor activity in pancreatic cancer cells by affecting multiple biochemical pathways.

ObjectivePancreatic cancer is one of the most lethal types of cancer with a 5-year survival rate of ~5%. Histone deacetylases (HDACs) participate in many cellular processes, including carcinogenesis, and pharmacological inhibition of HDACs has emerged as a potential therapeutic strategy. In this study, we explored antitumor activity of the novel HDAC inhibitor AR-42 in pancreatic cancer.MethodsHuman pancreatic cancer cell lines BxPC-3 and PANC-1 were used in this study. Real-time PCR, RT-PCR, and western blotting were employed to investigate expression of specific genes and proteins, respectively. Translocation of apoptosis-inducing factor was investigated by immunofluorescence and subcellular fractionation. The number of apoptotic cells, cell cycle stages, and reactive oxygen species (ROS) generation levels were determined by flow cytometry. Cell invasiveness was examined by the Matrigel invasion assay. Efficacy of AR-42 in vivo was evaluated by utilizing BxPC-3 xenograft mouse model.ResultsAR-42 inhibited pancreatic cancer cell proliferation by causing G2/M cell cycle arrest via regulating expression levels of genes and proteins involved in cell cycle. AR-42 also induced ROS generation and DNA damage, triggering apoptosis of pancreatic cancer cells via both caspase-3-dependent and caspase-3-independent pathways. In addition, AR-42 increased expression levels of negative regulators of p53 (miR-125b, miR-30d, and miR33), which could contribute to lower expression level of mutant p53 in pancreatic cancer cells. Cell invasion assay showed that AR-42 reduced cancer cell aggressiveness and significantly diminished BxPC-3 xenograft tumor growth in vivo.ConclusionAR-42, a novel HDAC inhibitor, inhibited pancreatic cancer cells by regulating p53 expression, inducing cell cycle arrest, particularly at the G2/M stage, and activating multiple apoptosis pathways. Additionally, AR-42 inhibited cell invasiveness and potently suppressed pancreatic cancer tumors in vivo. We conclude that by virtue of its multiple mechanisms of action, AR-42 possesses a considerable potential as an antitumor agent in pancreatic cancer.

Lysosomal dysfunction and autophagy blockade contribute to IMB-6G-induced apoptosis in pancreatic cancer cells

Targeting the autophagic pathway is currently regarded as an attractive strategy for cancer drug discovery. Our previous work showed that IMB-6G is a novel N-substituted sophoridinic acid derivative with potent cytotoxicity against tumor cells, yet the effect of IMB-6G on autophagy and pancreatic cancer cell death remains unknown. Here, we show that IMB-6G inhibits the growth of MiaPaCa-2 and HupT-3 pancreatic cancer cells and induces caspase-mediated apoptosis, which is correlated with an accumulation of autophagic vacuoles. IMB-6G promotes autophagosome accumulation from the early stage of treatment but blocks autophagic flux in the degradation stage, mainly through attenuation of lysosomal cathepsin activity in pancreatic cancer cells. Moreover, IMB-6G triggers lysosomal membrane permeabilization (LMP), followed by cathepsin B/CTSB and cathepsin D/CTSD release from lysosomes into the cytoplasm. Inhibition of autophagosome formation with siRNA against autophagy protein 5 (Atg5) attenuates IMB-6G-induced LMP and apoptosis. Furthermore, cathepsin inhibitors relieve IMB-6G-induced apoptosis as well. Altogether, our findings demonstrate that IMB-6G is a novel autophagy inhibitor, which induces autophagy-dependent apoptosis through autophagosomal-cathepsin axis in pancreatic cancer cells and indicate the potential value of IMB-6G as a novel antitumor drug candidate.

MicroRNA-296 targets AKT2 in pancreatic cancer and functions as a potential tumor suppressor

Although microRNA-296 (miR-296) has been studied in various types of human cancer, its expression, biological role and mechanism of action in pancreatic cancer remains to be elucidated. The aim of the current study was to investigate the expression level, possible roles and underlying molecular mechanisms of miR‑296 in pancreatic cancer. The present study revealed that miR‑296 is significantly downregulated in tissue from patients with pancreatic cancer and in human pancreatic carcinoma cell lines, when compared with matched healthy tissue and normal human pancreatic cell lines, respectively. In addition, restoration of miR‑296 expression was revealed to inhibit the proliferation, migration and invasive activity of pancreatic cancer cells. Furthermore, bioinformatics analysis and a luciferase reporter assay validated the AKT2 gene as a direct target of miR‑296 in pancreatic cancer. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis revealed that miR‑296 was able to decrease AKT2 expression at the post‑transcriptional level. Notably, the effects of AKT2 knockdown were similar to miR‑296 overexpression in pancreatic cancer. In conclusion, the present findings indicate a role for miR‑296 as a tumor suppressor in pancreatic cancer through directly targeting AKT2, thus suggesting that miR‑296 may serve as a potential therapeutic target for the treatment of pancreatic cancer.

Abstract C22: ROCK kinases drive invasive pancreatic tumor growth

Abstract Pancreatic cancer is one of the leading causes of cancer death and despite advances in chemotherapeutic regimens the overall 5-year survival rate remains less than 5%. The actomyosin-regulating ROCK1 and ROCK2 kinases are downstream-targets of the Rho GTPase pathway. They contribute to various processes, such as cell adhesion, motility, proliferation, differentiation and survival, which then influence numerous stages of cancer growth and progression. Interestingly, exome sequencing of pancreatic cancer genomes revealed that 15% of pancreatic cancer patients carry an amplification of the ROCK1 gene (1). Moreover, we found significant increases in ROCK1 and ROCK2 RNA expression in pancreatic cancer datasets obtained using Oncomine. When we further determined the protein expression of ROCK2 in pancreatic ductal adenocarcinoma (PDAC) we found an up-regulation of ROCK2 during disease progression. Higher ROCK2 levels also correlated with less differentiated tumors. A characteristic of advanced stages of pancreatic cancer is a collagen and fibroblast enriched stroma. To analyze the effect of ROCK kinases on cancer cell invasion, we performed 3-dimensional organotypic assays. Our results demonstrate that increased ROCK signaling was sufficient to convert non-invasive PDAC cells into ones capable of invasion into organotypic collagen matrix. Using an RNA sequencing approach, we investigated the effect of ROCK activation on pancreatic cancer cell gene transcription. Differential expression analysis revealed an enrichment of gene sets that are involved in cell-matrix interaction, and we found a higher release of matrix metalloproteinases MMP10 and MMP13 upon ROCK activation. In addition, our organotypic studies revealed extensive tissue remodeling and an accumulation of cleaved collagen bundles at the sites of PDAC cell invasion. Furthermore, collagen degradation and cell invasion into organotypic matrices were significantly reduced by application of a broad-spectrum MMP inhibitor, confirming that ROCK-induced invasion is dependent on MMP activity. Interestingly, our studies also indicated that ROCK-driven invasion of PDAC cells into collagen matrix enabled cell growth. To study the function of ROCK kinase signaling in pancreatic cancer in vivo, we expressed conditionally active ROCK2 in a mouse model of PDAC. We found that an increase of ROCK activity in pancreatic cancer cells accelerated PDAC progression, which resulted in reduced survival. Contrary, the administration of a ROCK inhibitor during tumor progression had a beneficial effect on survival. In summary, our results suggest that targeting ROCK kinases should be considered for chemotherapy of invasive pancreatic cancer. (1) Biankin et al. Nature. 2012 Nov 15;491(7424):399-405 Citation Format: Nicola Rath, Shereen Kadir, Jennifer P. Morton, Andreia V. Pinho, Lena Helbig, Linda Julian, Ewan J. McGhee, Gabriela Kalna, Alexei Vazquez, Kurt I. Anderson, Ilse Rooman, Michael S. Samuel, Michael F. Olson. ROCK kinases drive invasive pancreatic tumor growth. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C22.

Abstract 3061: AS-10: a new small molecule with promising activity against pancreatic cancer

Abstract Patients with pancreatic cancer (PC) have a median survival of only 6 months and a five-year survival of less than 5%, hence making PC one of the deadliest cancer. Severity of PC is due to its identification at late stages, rapid local invasion, early metastases, and meager response to current chemotherapeutic agents. Current therapies result in minimal survival advantage and are linked with multiple adverse events and drug resistance. Hence, there is an urgent need for novel agents which are less toxic and offer greater benefits over conventional therapy. There is ample evidences in literature demonstrating that inflammation plays a critical role in PC growth and promotion. Nuclear factor κB (NF-κB) pathway, one of the major inflammatory pathway, is well known for its inflammatory response, cell proliferation, and resistance to apoptosis. It has been demonstrated that activation of NF-κB in PC is also responsible for resistance towards first line chemotherapeutic agent, gemcitabine, in PC. Through extensive structure-activity relationship studies, we have recently identified a novel small molecule, AS-10, which was lethal to PC cells. We sought to evaluate the mechanism of action of AS-10 responsible for inhibiting the growth of PC cells. In vitro, AS-10 reduced PC cell growth with an EC50, in the range of 2.5 to 5 μM. Growth arrest was confirmed by cell cycle studies, which showed that AS-10 induced G1 and G2 cell cycle arrest. The cell cycle arrest was associated with an increase of cell cycle inhibitory markers like p21 and p27. Effect of AS-10 on cell cycle was translated into activation of apoptosis, confirmed by caspase 3/7 activity, PARP cleavage and Annexin V staining. Due to its structural characteristics, we evaluated the effect of AS-10 on inflammatory NF-κB pathway. Our studies, in Panc-1 cells, showed that AS-10 inhibited NF-κB DNA binding activity as well as NF-κB translocation to the nuclei in the presence of inflammatory stimuli (tumor necrosis factor (TNFα)). Since, up regulation of NF-κB activity has been known to be responsible for gemcitabine resistance in PC, we evaluated the activity of our NF-κB inhibitor AS-10 in combination with gemcitabine, and showed that AS-10 synergistically potentiated gemcitabine activity in PC cells. Taken together, these results suggest that AS-10, may represent a potentially promising therapeutic agent for PC. Detailed investigations regarding the efficacy and mechanism of action of these compounds will be presented. Citation Format: Deepkamal Karelia, Manoj K. Pandey, Daniel Plano, Shantu Amin, Arun K. Sharma. AS-10: a new small molecule with promising activity against pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3061.

Resveratrol inhibits hyperglycemia-driven ROS-induced invasion and migration of pancreatic cancer cells via suppression of the ERK and p38 MAPK signaling pathways

Increasing evidence suggests that there is a strong relationship between diabetes mellitus (DM) and pancreatic cancer. Our previous study revealed that hyperglycemia could enhance the invasive and migratory activities of pancreatic cancer cells. Resveratrol, a natural polyphenolic phytoalexin, has many biological and pharmaceutical properties, including antioxidant and anti-tumorigenic capabilities. The aim of the present study was to evaluate whether resveratrol affects hyperglycemia-induced reactive oxygen species (ROS) production as well as the invasion and migration of pancreatic cancer and its underlying mechanisms. Human pancreatic cancer Panc-1 cells were exposed to high glucose condition with or without resveratrol, N-acetylcysteine (NAC, a scavenger of free radicals), PD 98059 (an ERK inhibitor) or SB 203580 (a p38 MAPK inhibitor). The intracellular ROS and hydrogen peroxide (H2O2) were determined using 2,7-dichlorodihydrofluorecein diacetate and H2O2 assay. MTT, wound healing assay and transwell matrigel invasion assay were used to detect the proliferation, migration and invasion potential of cancer cells. The expressions of uPA, E-cadherin and Glut-1 were examined using QT-PCR and western blot analysis at mRNA and protein levels. The activation of p-ERK, p-p38 and p-NF-κB were measured by western blot analysis. The results of the present study showed that resveratrol could significantly decrease high glucose-induced production of ROS and H2O2 in Panc-1 cells. Resveratrol was also able to inhibit high glucose-induced proliferation, migration and invasion of pancreatic cancer cells. High glucose-modulated expression of uPA, E-cadherin and Glut-1 were inhibited by resveratrol. In addition, high glucose-induced activation of ERK and p38 MAPK signaling pathways as well as the transcription factor NF-κB could also be suppressed by resveratrol. Furthermore, resveratrol was able to suppress H2O2-induced migration and invasion abilities of pancreatic cancer cells. Taken together, these data indicate that resveratrol plays an important role in suppressing hyperglycemia-driven ROS-induced pancreatic cancer progression by inhibiting the ERK and p38 MAPK signaling pathways, providing evidence that resveratrol might be a potential candidate for chemoprevention of pancreatic cancer.

Panobinostat as Pan-deacetylase Inhibitor for the Treatment of Pancreatic Cancer: Recent Progress and Future Prospects.

The histone deacetylase (HDAC) inhibitors have been demonstrated as an emerging class of anticancer drugs. HDACs are involved in regulation of gene expression and in chromatin remodeling, thus indicating valid targets for different types of cancer therapeutics. The pan-deacetylase inhibitor panobinostat (Farydac®, LBH589) was developed by Novartis Pharmaceuticals and has been recently approved by the US Food and Drug Administraion (FDA) as a drug to treat multiple myeloma. It is under clinical investigation for a range of haematological and solid tumors worldwide in both oral and intravenous formulations. Panobinostat inhibits tumor cell growth by interacting with acetylation of histones and non-histone proteins as well as various apoptotic, autophagy-mediated targets and various tumorogenesis pathways involved in development of tumors. The optimal combination regimen for pancreatic cancer remains to be fully elucidated with various combination regimens, and should be investigated in clinical trials. This article summarizes the current preclinical and clinical status of panobinostat in pancreatic cancer. Preclinical data suggests that panobinostat has potential inhibitory activity in pancreatic cancer cells by targeting various pathways and factors involved in the development of cancer. Herein, we reviewed the status of mono and combination therapy and the rationale behind the combination therapy undergoing trials, as well as possible future prospective use in the treatment of pancreatic cancer.

Downregulation of STAT3/NF-κB potentiates gemcitabine activity in pancreatic cancer cells.

There is an unmet need to develop new agents or strategies against therapy resistant pancreatic cancer (PanCA). Recent studies from our laboratory showed that STAT3 negatively regulates NF-κB and that inhibition of this crosstalk using Nexrutine® (Nx) reduces transcriptional activity of COX-2. Inhibition of these molecular interactions impedes pancreatic cancer cell growth as well as reduces fibrosis in a preclinical animal model. Nx is an extract derived from the bark of Phellodendron amurense and has been utilized in traditional Chinese medicine as antidiarrheal, astringent, and anti-inflammatory agent for centuries. We hypothesized that "Nx-mediated inhibition of survival molecules like STAT3 and NF-κB in pancreatic cancer cells will improve the efficacy of the conventional chemotherapeutic agent, gemcitabine (GEM)." Therefore, we explored the utility of Nx, one of its active constituents berberine and its derivatives, to enhance the effects of GEM. Using multiple human pancreatic cancer cells we found that combination treatment with Nx and GEM resulted in significant alterations of proteins in the STAT3/NF-κB signaling axis culminating in growth inhibition in a synergistic manner. Furthermore, GEM resistant cells were more sensitive to Nx treatment than their parental GEM-sensitive cells. Interestingly, although berberine, the Nx active component used, and its derivatives were biologically active in GEM sensitive cells they did not potentiate GEM activity when used in combination. Taken together, these results suggest that the natural extract, Nx, but not its active component, berberine, has the potential to improve GEM sensitivity, perhaps by down regulating STAT3/NF-κB signaling. © 2016 Wiley Periodicals, Inc.

Abstract C138: Targeting macropinocytosis in pancreatic cancer

Abstract Pancreatic cancer is the fourth-leading cause of cancer-related deaths in the United States with a 5-year survival rate of <5%. The deadly nature of this disease compounded by a lack of effective treatment options highlights a need for new therapeutics. Past studies have suggested that pancreatic cancer cells acquire the nutrients needed for cell growth through macropinocytosis, an endocytic process by which protrusions from the cell membrane create large vesicles that transport extracellular fluid along with the constituent proteins and lipids into the cell. Additionally, several studies have suggested that macropinocytosis is regulated in part by various genes including p21 protein (Cdc42/Rac)-activated kinase1 (PAK1), ADP-ribosylation factor 6 (ARF6), and sorting nexin 5 (SNX5). In this study, we investigated the effects of knockdown of these three genes on the macropinocytic activity of pancreatic cancer cells. The goal of this study is to assess whether PAK1, ARF6, or SNX5 may serve as potential targets for the suppression of macropinocytosis in pancreatic cancer, which would hamper the cells' uptake of nutrients thus starving the cancer cells and hindering tumor growth. We carried out this study using MIA PaCa-2 pancreatic cancer cells, which are known to exhibit relatively high levels of macropinocytosis. Treatment of cells with siRNA sequences specific to the three genes resulted in a significantly decreased uptake of FITC-labeled dextran as compared to cells treated with non-targeting siRNA and cells that received no treatment, indicating that inhibiting the expression of each of the genes correlates to a reduction in macropinocytic activity. Among the three genes, PAK1 siRNAs exhibited the most significant and consistent inhibitory effect on macropinocytic activity of MIA PaCa-2 cells. We, therefore, further tested the effect of two small-molecule PAK1 inhibitors, namely IPA-3 and PF-03758309, on macropinocytosis. Treatment of cells with the inhibitors resulted in a dose-dependent decrease in the uptake of FITC-labeled dextran and FITC-labeled albumin. The inhibitors also showed potent anti-proliferative activity in pancreatic cancer cell lines. The results of this study indicate that inhibition of PAK1, ARF6, or SNX5 all resulted in reduced macropinocytic activity of pancreatic cancer cells, with PAK1 showing the most promise as a potential therapeutic target for pancreatic cancer. Citation Format: James Hunt, Serina Ng, Clifford Whatcott, Daniel D. Von Hoff, Haiyong Han. Targeting macropinocytosis in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C138.

Abstract A63: Role of the metastasis suppressor, NDRG1, in regulating the EGFR and ErbB family of receptors and its effects on key oncogenic signaling pathways in pancreatic cancer

Abstract N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that has been shown to affect numerous signaling pathways that control oncogenesis. In this study, the role of NDRG1 was investigated on a key upstream effector, namely epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor 2 (HER2) and human epidermal growth factor receptor 3 (HER3). This is of interest, as the ErbB family of receptor tyrosine kinases are involved in regulating multiple cell responses, being key regulators of down-stream oncogenic-signaling. We demonstrate that NDRG1 is able to significantly reduce the expression, localisation and activation of EGFR, HER2 and HER3, while also inhibiting the formation of EGFR/HER2 and HER2/HER3 heterodimers. This investigation also showed that a novel class of anti-cancer agents, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), were able to markedly up-regulate NDRG1. These agents were found to inhibit EGFR, HER2 and HER3 expression and phosphorylation in PANC-1 pancreatic cancer cells in vitro. Moreover, these compounds led to a significant reduction of the expression of these proteins in PANC-1 tumour xenografts in vivo. Due to the limitations of current anti-cancer therapeutics, these agents were also compared to a clinically used EGFR inhibitor, Erlotinib. Our study showed that in comparison to Erlotinib, both Dp44mT and DpC displayed higher anti-proliferative activity in pancreatic cancer cells. This could be significant, as DpC is due to enter clinical trials this year for the treatment of aggressive solid tumours (http://www.colmeddev.com/oncochel/). Together, these findings reveal the molecular mechanisms that underlie the anti-cancer effects of NDRG1, and in turn, demonstrate the potential origin of the extensive down-stream effects attributed to this molecule. Citation Format: Sharleen V. Menezes, Zaklina Kovacevic, Des R. Richardson. Role of the metastasis suppressor, NDRG1, in regulating the EGFR and ErbB family of receptors and its effects on key oncogenic signaling pathways in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A63.

CK1δ activity is modulated by CDK2/E- and CDK5/p35-mediated phosphorylation.

CK1 protein kinases form a family of serine/threonine kinases which are highly conserved through different species and ubiquitously expressed. CK1 family members can phosphorylate numerous substrates thereby regulating different biological processes including membrane trafficking, cell cycle regulation, circadian rhythm, apoptosis, and signal transduction. Deregulation of CK1 activity and/or expression contributes to the development of neurological diseases and cancer. Therefore, CK1 became an interesting target for drug development and it is relevant to further understand the mechanisms of its regulation. In the present study, Cyclin-dependent kinase 2/Cyclin E (CDK2/E) and Cyclin-dependent kinase 5/p35 (CDK5/p35) were identified as cellular kinases able to modulate CK1δ activity through site-specific phosphorylation of its C-terminal domain. Furthermore, pre-incubation of CK1δ with CDK2/E or CDK5/p35 reduces CK1δ activity in vitro, indicating a functional impact of the interaction between CK1δ and CDK/cyclin complexes. Interestingly, inhibition of Cyclin-dependent kinases by Dinaciclib increases CK1δ activity in pancreatic cancer cells. In summary, these results suggest that CK1δ activity can be modulated by the interplay between CK1δ and CDK2/E or CDK5/p35. These findings extend our knowledge about CK1δ regulation and may be of use for future development of CK1-related therapeutic strategies in the treatment of neurological diseases or cancer.

Abstract 670: Targeting PI3K/mTOR leads to MEK/ERK over-activation in pancreatic cancer through suppression of mTORC2

Abstract Significant attention has been given to molecular target therapies in pancreatic cancer due to the limited survival benefits provided by standard chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1/S6K pathway which is aberrantly stimulated in pancreatic ductal adenocarcinoma (PDAC) has emerged as a target for therapy. Inhibitors of PI3K and/or mTOR (PI3K/TOR-KIs) are been developed to target this pathway. However, mTORC1/S6K also mediates negative feedback loops that attenuate upstream signaling. Suppression of these feedback loops opposes the growth-suppressive effects of mTOR inhibitors and leads to drug resistance. Here, we demonstrate that treatment of a panel of pancreatic ductal adenocarcinoma (PDAC) cells including PANC-1, MiaPaCa-2, AsPC-1 and BxPC-3 with the dual PI3K/mTOR kinase inhibitor (PI3K/TOR-KI) NPV-BEZ23 induces a novel negative feedback loop leading to increased activity of MEK/ERK pathway in PDAC cells. Exposure of the above mentioned PDAC cells to NPV-BEZ235 potently blocked mTORC1 activation mTORC1/S6K activation (scored by S6 phosphorylation at Ser240/244), mTORC1/4E-BP1 (assayed by 4E-BP1 phosphorylation at Thr37/46) and mTORC2-mediated Akt phosphorylation at Ser473, in a concentration-dependent manner. Strikingly, NPV-BEZ235 markedly enhanced the MEK/ERK pathway in a dose-dependent manner. Maximal ERK over-activation coincided with complete inhibition of phosphorylation of Akt and 4E-BP1. Other PI3K/TOR-KIs, including PKI-587 and GDC-0980 also induced ERK over-activation. The MEK inhibitors PD0325901 or Trametinib prevented ERK over-activation induced by PI3K/TOR-KIs at least acutely. The combination of NPV-BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor individually. Mechanistic studies assessing PI3K activity in single PDAC cells using a PIP3 fluorescent reporter indicated that NPV-BEZ235, PKI-587 and GDC-0980 act through a PI3K-independent pathway. Doses of PI3K/TOR-KIs that enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated Akt phosphorylation on Ser473, suggesting a role of mTORC2. Knockdown of Rictor markedly increased baseline levels of ERK phosphorylation and treatment with NVP-BEZ235 did not produce further enhancement of ERK activation. These results imply that Rictor or mTORC2 exerts feedback inhibition of the MEK/ERK pathway in pancreatic cancer cells. We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2 thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells. The therapeutic effectiveness of PI3K/TOR-KIs inhibitors in PDAC and possibly other malignancies can be diminished by activation of MEK/ERK that opposes their anti-proliferative effects and leads to drug resistance. Note: This abstract was not presented at the meeting. Citation Format: Heloisa P. Soares, Ming Ming, Michelle Mellon, Steven H. Young, Liang Han, James Sinnet-Smith, Enrique Rozengurt. Targeting PI3K/mTOR leads to MEK/ERK over-activation in pancreatic cancer through suppression of mTORC2. [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 670. doi:10.1158/1538-7445.AM2015-670

Abstract A89: Targeting the MYC activation and degradation pathway for the treatment of pancreatic cancer

Abstract Pancreatic cancer is a disease with a high rate of mortality, as it is generally diagnosed in the advanced stages when few effective treatments are available. Given the clinical aggressivity of this disease and lack of good treatment options, the development of new therapeutic agents is of the upmost importance. The c-MYC (MYC) oncoprotein is a critical transcription factor that regulates many diverse cellular functions by either activating or suppressing downstream target genes. High levels of MYC expression occur in a wide variety of human tumors, and animal models demonstrate MYC-induced tumorigenesis in many tissues. It has been shown that the MYC gene is frequently amplified in human pancreatic cancer and that MYC mRNA and/or protein are often overexpressed. However, what pathways contribute to this overexpression or whether MYC can be therapeutically targeted in pancreatic cancer is yet not known. Work in our laboratory has elucidated a complex signaling pathway that regulates MYC protein stability and transcriptional activity. RAS activation in early G1 leads to ERK-dependent phosphorylation of MYC at Serine62 (pS62-MYC), which stabilizes MYC. pS62-MYC is then recognized by the phosphorylation-directed Pin1 prolyl isomerase, which isomerizes Proline63 (P63) to cis, enhancing MYC’s recruitment to, and transactivation of, pro-growth target genes. Later in G1, when RAS activation decreases, GSK3β phosphorylates MYC at T58 (pT58-MYC). Following a second isomerization by Pin1, the trans-specific phosphatase PP2A-B56α removes the stabilizing S62 phosphate. pT58-MYC is then recognized by the E3 ubiquitin ligase SCFFbw7 and degraded by the proteasome. We have found that this pathway is frequently deregulated in human cancer leading to elevated pS62, decreased pT58, and increased c-Myc stability and activity. Here, we show that MYC is overexpressed and stabilized in pancreatic cancer cells. Additionally, we find aberrant MYC phosphorylation in primary human pancreatic cancer tissue, suggesting that the pathway that controls MYC phosphorylation is deregulated in human pancreatic cancer. Therefore, we hypothesized that the pathways regulating MYC phosphorylation and activation might represent new therapeutic targets. We are pursuing two different approaches to target MYC in pancreatic cancer: 1) inhibiting Pin1, the prolyl isomerase that co-activates pS62-MYC, and 2) activation of PP2A, the phosphatase that destabilizes MYC protein. We have observed decreased PP2A activity in pancreatic cancer cells, and have found that two endogenous inhibitors of PP2A, SET and CIP2A, are frequently overexpressed in pancreatic cancer cells and primary patient samples. This overexpression correlates with aberrant phosphorylation and stabilization of MYC. Furthermore, knockdown of SET or CIP2A, or inhibition of SET using a peptide mimetic (OP449), increases PP2A activity and reduces the tumorigenic potential of pancreatic cancer cells both in vitro and in vivo. In addition to increased SET and CIP2A expression in pancreatic cancer, we also observe frequent overexpression of Pin1 in human pancreatic cancer cell lines and primary patient tumor tissue and find that Pin1 knockdown or inhibition reduces the oncogenic potential of pancreatic cancer cells both in vitro and in vivo. Together, these findings suggest that MYC may play an important role in pancreatic cancer, and that targeting the pathway that regulates MYC activity and protein stability may be a viable therapeutic approach for human pancreatic cancer. Citation Format: Amy S. Farrell, Brittany Allen-Petersen, Colin J. Daniel, Xiaoyan Wang, Zhiping Wang, Dale J. Christensen, Brett Sheppard, Rosalie C. Sears. Targeting the MYC activation and degradation pathway for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A89.

Abstract A104: Phosphatase activation and kinase inhibition as a novel therapeutic strategy for pancreatic cancer

Abstract The 5-year survival rate for pancreatic cancer patients is only 6%, the lowest of all major cancers, indicating a critical need for increased understanding of pancreatic cancer development and identification of new therapeutic targets. Several genetic mutations are associated with the progression of human pancreatic ductal adenocarcinoma, including KRAS, which is mutated in ~95% of all pancreatic ductal adenocarcinomas (PDAC). Targeted therapies, such as kinase inhibitors, provide a means to reduce oncogenic signaling pathways that drive pancreatic cancer; however, the presence of resistance mechanisms, through extensive feedback loops, reduces the clinical efficacy of single agent treatments. Protein Phosphatase 2A (PP2A) is a critical tumor suppressor that negatively regulates several key oncogenic pathways implicated in mediating therapeutic resistance, including the PI3K/Akt, RAS/ERK, and Myc pathways. The goal of this research is to identify the therapeutic benefit of combined phosphatase activation and kinase inhibition, as a means to attenuate resistance mechanisms, reduce pancreatic tumor growth, and improve therapeutic efficacy in pancreatic cancer patients. We have determined that SET, an endogenous inhibitor of PP2A, is overexpressed in pancreatic cancer cell lines and primary patient samples, suggesting that suppression of PP2A contributes to pancreatic oncogenic signaling. Therapeutic inhibition of SET utilizing a novel SET antagonist, OP449, leads to increased PP2A activity and significant cytotoxic activity in pancreatic cancer cells in vitro and in vivo. Importantly, these results correlate with reduced activation of pathways known to contribute to therapeutic resistance, including c-Myc and Akt. I hypothesize that phosphatase activation will function synergistically with select kinase inhibitors, resulting in sustained attenuation of oncogenic signaling in pancreatic cancer cells. In order to identify pathways that function synergistically with PP2A activation, a panel of pancreatic cancer cell lines was plated with and without OP449 into a 384-well kinase inhibitor (KI) screen, which evaluates the efficacy of over 130 kinase inhibitors at seven serial dilutions spanning the predicted IC50. Several signaling nodes were found to be synergistic with OP449, with the PI3K/AKT/mTOR pathways being particularly susceptible. Specifically, the combination of OP449 with Ink128, an mTOR1/2 inhibitor, reduced pancreatic cancer cell survival, oncogenic signaling, and transformed phenotypes, over either drug alone. These results provide new insight into our understanding of the regulation of pancreatic oncogenic signaling by phosphatases and identify new combination therapies that reduce resistance mechanisms. Citation Format: Brittany L. Allen-Petersen, Amy S. Farrell, Colin J. Daniel, Zhiping Wang, Charles D. Lopez, Dale J. Christensen, Brett C. Sheppard, Rosalie C. Sears. Phosphatase activation and kinase inhibition as a novel therapeutic strategy for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A104.

Quercetin 3-O-glucoside suppresses epidermal growth factor-induced migration by inhibiting EGFR signaling in pancreatic cancer cells.

Pancreatic cancer is one of the most dangerous cancers and is associated with a grave prognosis. Despite increased knowledge of the complex signaling networks responsible for progression of pancreatic cancer, many challenging therapies have fallen short of expectations. In this study, we examined the anti-migratory effect of quercetin 3-O-glucoside in epidermal growth factor-induced cell migration by inhibiting EGF receptor (EGFR) signaling in several human pancreatic cancer cell lines. Treatment with quercetin, quercetin 3-O-glucoside, and quercetin 7-O-glucoside differentially suppressed epidermal growth factor-induced migration activity of human pancreatic cancer cells. In particular, quercetin 3-O-glucoside strongly inhibited the infiltration activity of pancreatic cancer cells in a dose-dependent manner. Furthermore, quercetin 3-O-glucoside exerted the anti-migratory effect even at a relatively low dose compared with other forms of quercetin. The anti-tumor effects of quercetin 3-O-glucoside were mediated by selectively inhibiting the EGFR-mediated FAK, AKT, MEK1/2, and ERK1/2 signaling pathway. Combinatorial treatment with quercetin 3-O-glucoside plus gemcitabine showed the synergistic anti-migratory effect on epidermal growth factor-induced cell migration in human pancreatic cancer cell lines. These results suggest that quercetin 3-O-glucoside has potential for anti-metastatic therapy in human pancreatic cancer.

Twist promotes invasion and cisplatin resistance in pancreatic cancer cells through growth differentiation factor 15.

Pancreatic cancer (PC) is an aggressive and devastating disease with a poor prognosis. Cisplatin, a commonly used chemotherapeutic agent for solid tumors, is effective as a single agent or in combination with other drugs for the treatment of PC. Previous studies have suggested that Twist and growth differentiation factor 15 (GDF15) are involved in the progression of PC. However, the role of Twist and GDF15 in PC remains to be elucidated. In the present study, the individual effect of and interaction between Twist and GDF15 in PC cell invasion and chemoresistance to cisplatin was examined. Twist and/or GDF15 were stably overexpressed or knocked down in ASPC‑1 and BXPC‑3 human PC cells. Overexpression of Twist in the two cell lines markedly increased GDF15 expression, cell invasion, matrix metalloproteinase‑2 expression/activity and the half maximal inhibitory concentration (IC50) values of cisplatin, which was eradicated by GDF15 knockdown or the selective p38 mitogen‑activated protein kinase (MAPK) inhibitor SB203580 (10 µM). By contrast, Twist knockdown significantly decreased GDF15 expression, cell invasion, matrix metalloproteinase‑2 expression/activity and the IC50 values of cisplatin, which was completely reversed by overexpression of GDF15. In addition, while overexpression and knockdown of Twist increased and decreased p38 MAPK activity, respectively, GDF15 demonstrated no significant effect on p38 MAPK activity in PC cells. In conclusion, the present study, for the first time, to the best of our knowledge, demonstrated that Twist promotes PC cell invasion and cisplatin chemoresistance through inducing GDF15 expression via a p38 MAPK‑dependent mechanism. The present study provides new insights into the molecular mechanisms underlying PC progression and chemoresistance.

Expression and Activity of Nadph Oxidase 4 (Nox4) is Related to Antiapoptotic Activity of Human Pancreatic Adenocarcinoma Cells

Pancreatic adenocarcinoma is one of the most aggressive human malignancies with high mortality rates. Low survival rates are due to late diagnosis at advanced stages of the disease. High invasiveness and chemoresistance of pancreatic adenocarcinoma at least partially could be related to the antiapoptotic activity of intracelular ROS generated by Nox4. There are only few studies about Nox4 expression in pancreatic cancer, yet Nox4 is believed to be relevant antiapoptotic factor in pancreatic cancer cells. In this study we have determined the expression of Nox4 in human pancreatic tissue at both protein and mRNA levels. We compared how Nox4 is overexpressed in pancreatic cancer in comparison to human healthy adjacent and healthy donor pancreatic tissue. We have also identified the effect of ROS formation in MiaPaca-2 and Capan-2 cells under treatment with NADPH oxidase inhibitors DPI and apocynin. Our results showed that DPI and apocynin inhibited ROS formation. Moreover, it decreased viability of pancreatic cancer cells and induced apoptosis as well. Result confirms ROS being responsible for antiapoptotic activity of pancreatic cancer cells. These findings point out to NADPH oxidases being a potential terapeutic targets in treatment of cancer.

Dual PI3K/mTOR Inhibitors Induce Rapid Overactivation of the MEK/ERK Pathway in Human Pancreatic Cancer Cells through Suppression of mTORC2.

The PI3K/AKT/mTOR pathway, which is aberrantly stimulated in many cancer cells, has emerged as a target for therapy. However, mTORC1/S6K also mediates negative feedback loops that attenuate upstream signaling. Suppression of these feedback loops opposes the growth-suppressive effects of mTOR inhibitors and leads to drug resistance. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic ductal adenocarcinoma (PDAC) cells with the dual PI3K/mTOR kinase inhibitor (PI3K/TOR-KI) BEZ235 blocked mTORC1/S6K activation (scored by S6 phosphorylation at Ser(240/244)), mTORC1/4E-BP1 (assayed by 4E-BP1 phosphorylation at Thr(37/46)), and mTORC2-mediated AKT phosphorylation at Ser(473), in a concentration-dependent manner. Strikingly, BEZ235 markedly enhanced the MEK/ERK pathway in a dose-dependent manner. Maximal ERK overactivation coincided with complete inhibition of phosphorylation of AKT and 4E-BP1. ERK overactivation was induced by other PI3K/TOR-KIs, including PKI-587 and GDC-0980. The MEK inhibitors U126 or PD0325901 prevented ERK overactivation induced by PI3K/TOR-KIs. The combination of BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor individually. Mechanistic studies assessing PI3K activity in single PDAC cells indicate that PI3K/TOR-KIs act through a PI3K-independent pathway. Doses of PI3K/TOR-KIs that enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated AKT phosphorylation on Ser(473), suggesting a role of mTORC2. Knockdown of RICTOR via transfection of siRNA markedly attenuated the enhancing effect of BEZ235 on ERK phosphorylation. We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2, thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.

Tu1973 The Novel Observation of Irreversible Electroporation Phenomenon on Gastrointestinal Cancer Cells in Physiological Conditions, With Sub-Micrometer Resolution by Scanning Ion Conductance Microscopy

G A A b st ra ct s enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated Akt phosphorylation on Ser473, suggesting a role of mTORC2. Knockdown of Rictor markedly increased baseline levels of ERK phosphorylation and treatment with NVP-BEZ235 did not produce further enhancement of ERK activation. These results imply that Rictor or mTORC2 exerts feedback inhibition of the MEK/ERK pathway in pancreatic cancer cells. Conclusion: We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2 thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.