Green synthesis of gold nanoparticles using Acai berry and Elderberry extracts and investigation of their effect on prostate and pancreatic cancer cells.

Desmoplasia of Pancreatic Ductal Adenocarcinoma

The aminobisphosphonate zoledronic acid has elicited significant attention due to its remarkable anti-tumoral activity, although its detailed mechanism of action remains unclear. Here, we demonstrate the existence of a nuclear GSK-3β-NFATc2 stabilization pathway that promotes breast and pancreatic cancer growth in vitro and in vivo and serves as a bona fide target of zoledronic acid. Specifically, the serine/threonine kinase GSK-3β stabilizes nuclear NFATc2 through phosphorylation of the serine-rich SP2 domain, thus protecting the transcription factor from E3-ubiquitin ligase HDM2-mediated proteolysis. Zoledronic acid disrupts this NFATc2 stabilization pathway through two mechanisms, namely GSK-3β inhibition and induction of HDM2 activity. Upon nuclear accumulation, HDM2 targets unphosphorylated NFATc2 for ubiquitination at acceptor lysine residues Lys-684/Lys-897 and hence labels the factor for subsequent proteasomal degradation. Conversely, mutagenesis-induced constitutive serine phosphorylation (Ser-215, Ser-219, and Ser-223) of the SP2 domain prevents NFATc2 from HDM2-mediated ubiquitination and degradation and consequently rescues cancer cells from growth suppression by zoledronic acid. In conclusion, this study demonstrates a critical role of the GSK-3β-HDM2 signaling loop in the regulation of NFATc2 protein stability and growth promotion and suggests that double targeting of this pathway is responsible, at least to a significant part, for the potent and reliable anti-tumoral effects of zoledronic acid.

Cell-cell interactions within the tumor microenvironment play critical roles in tumor progression and its response to various therapies. Specifically in the case of pancreatic cancer, pancreatic cancer cells (PCCs) act on pancreatic stellate cells (PSCs), a major stromal cell type within the pancreatic tumor microenvironment, to provoke their transformation into and sustain an activated state, while activated PSCs interact with PCCs to promote tumor progression and confer a favorable environment for PCC resistance to immunosuppression and therapeutic treatment, setting up a vicious cycle. Therefore, effectively attacking the stromal PSCs alone or together with tumor cells may offer a new therapeutic route for pancreatic ductal adenocarcinoma (PDA) treatment. To identify key signaling molecules or pathways activated as a result of PSC-PCC interactions that might be targeted in pancreatic cancer as novel therapies, we developed unbiased quantitative mass spectrometry (MS)-based proteomics strategies to perform systemic analysis. Our comprehensive molecular and cellular analysis of in vitro PSC and PSC co-culture unraveled many interesting secreted proteins, either known or unknown previously, involved in the paracrine PCC-PSC crosstalk. We mainly focused on an intriguing signaling loop, in which PCCs secrete PDGFs that act on PSCs to promote the production of inflammatory cytokines by PSCs, including IL-6 family members, and especially LIF. These cytokines then act on PCCs to stimulate STAT3 activation, a known critical event for PDA initiation and progression. Significantly increased production of the cytokines and expression of corresponding receptors in both mouse and human PDA tumor contexts at both mRNA and protein levels were confirmed using various techniques. To further evaluate the functional significance of LIF in paracrine regulation of PDA progression in vivo, we exploited therapeutic blockage of the signaling using neutralizing mAbs and specific small molecule inhibitors in the classical KPC mouse PDA model. Our preclinical studies revealed significant efficacy of an anti-LIF mAb in increasing sensitivity to the standard-of-care chemotherapeutic gemcitabine, reducing tumor progression and prolonging survival of KPC mice. Currently, the molecular mechanisms underlying this sensitization are being investigated. Genetic studies using LIF receptor conditional knockout mice were also performed and showed significant beneficial effects. Furthermore, potential correlations between tissue or serum LIF and IL-6 cytokine levels and overall survival of human patients are also being evaluated. Citation Format: Yu Shi, Ruijun Tian, Tony Hunter.{Authors}. Interdicting the cytokine-mediated paracrine communication between pancreatic cancer and stellate cells as a new treatment approach for pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B27.

Cancer is one of the most aggressive diseases whose prognosis remains bleak with current therapies. The tumor microenvironment plays a significant role in the proliferation and invasion of tumor cells. In the complex tumor microenvironment, there are several types of immune-suppressing infiltrating cells, which surround the tumor and promote tumor growth and metastasis. Prostate and pancreatic cancers display tumor-infiltrating immune cells, which differentiate into cells that promote each step of the metastatic cascade and therefore are considered as novel targets for therapy. Even though several chemotherapeutic drugs are in current use for treating these cancers, tumor microenvironment targeting immunotherapeutic drugs, with minimum systemic toxic effects, are needed to save human lives from various forms of cancers. We hypothesize a safe and effective nanoinitiated drug delivery system—‘Phytochemical-conjugated gold nanoparticles,’ which can inhibit the growth and proliferation of tumor cells without affecting normal cells and at the same time boost the capability of immune system to fight cancer. There are unprecedented opportunities to advance the treatment of cancer using nanotechnology. Among the vast majority of nanoparticles, gold nanoparticles show unique properties, such as large surface-to-volume ratio, small size in the nanorealm, tunable surface chemistry, and capable of drug encapsulation. The controlled and targeted drug delivery approach makes nanoparticles very selective and effective toward cancer growth inhibition. In this chapter, we discuss the synthesis and characterization of clove phytochemical-conjugated gold nanoparticles (CP-AuNPs) and their anticancer activity against prostate (PC-3) and pancreatic cancer (PANC-1) cell lines. The synthesis and full characterization of AuNPs were confirmed by various techniques including UV–Visible spectrophotometry, transmission electron microscopy (TEM), and dynamic light-scattering (DLS) technique. We confirmed that AuNP treatment resulted in decreased cancer cell viability and enhancement in cellular internalization. These nanoparticles will navigate better to the complex tumor sites. This ‘Green Nanotechnology’ approach will be potentially effective compared to the current conventional cancer therapies.

Epigenetic plasticity is a pivotal factor driving metastasis. Here, we show that the promoter of the gene encoding the ubiquitin ligase subunit FBXL7 is hypermethylated in advanced prostate and pancreatic cancers, correlating with decreased FBXL7 mRNA and protein levels. Low FBXL7 mRNA levels are predictive of poor survival in patients with pancreatic and prostatic cancers. FBXL7 mediates the ubiquitylation and proteasomal degradation of active c-SRC upon its phosphorylation on Ser104. The DNA-demethylating agent decitabine recovers FBXL7 expression and limits epithelial-to-mesenchymal transition and cell invasion in a c-SRC-dependent manner. In vivo, FBXL7-depleted cancer cells form tumors with high metastatic burden. Co-silencing of c-SRC or treatment with the c-SRC inhibitor dasatinib prevents metastases. Furthermore, decitabine reduces metastases derived from prostate and pancreatic cancer cells in a FBXL7-dependent manner. Collectively, this work implicates FBXL7 as a metastasis suppressor gene and suggests therapeutic strategies to counteract metastatic dissemination of pancreatic and prostatic cancer cells.

S2010 Purinergic P2y2 Receptor and CA 2+-Mediated Proliferation of Human Pancreatic Epithelial Cancer Cells

Targeting mitochondrial transcription factor A sensitizes pancreatic cancer cell to gemcitabine

Background: GPRC5A is an orphan G-protein coupled receptor acting as an oncogene in some cancers and as a tumor suppressor in other. Its role in pancreatic cancer has not been studied systematically. ELAV-like protein 1, also known as “HuR,” is an RNA binding protein that is encoded by the ELAVL1 gene. HuR post-transcriptionally regulates multiple genes, plays key roles in human malignancies, and is known to mediate the efficacy of gemcitabine. Our preliminary data indicated that, in pancreatic cells, GPRC5A transcription responded to gemcitabine treatment and that its mRNA likely interacted with HuR. Methods: We immunohistochemically analyzed GPRC5A protein expression in primary and metastatic pancreatic cancer samples. We examined the effects of GPRC5A on pancreatic cells’ ability to proliferate, migrate, and invade, and how gemcitabine modulates this ability. We also studied the interaction between GPRC5A and HuR and examined how it is affected by gemcitabine. Lastly, we used microarray expression analyses to identify the genes and pathways linked to GPRC5A in pancreatic cancer cells. Results: We analyzed public mRNA expression data from 675 human cancer cell lines and 10,609 samples from The Cancer Genome Atlas (TCGA): GPRC5A mRNA abundance in pancreatic cancer was the highest (cell lines) or second highest (TCGA) among all tissues and cancer types. We analyzed an independent set of 252 pancreatic normal and cancer samples: we found GPRC5A mRNA to be up-regulated in primary tumor samples compared to normal pancreas, and even further up-regulated in metastatic pancreatic cancer. We immunostained 208 cores (103 primary and metastatic pancreatic cancer samples): we found GPRC5A protein to be barely expressed in normal pancreatic ductal cells and highly expressed in both primary and metastatic pancreatic ductal cancer cells. We carried out in vitro studies of multiple pancreatic cancer cell lines: we found that increasing GPRC5A protein levels promoted pancreatic cancer cell growth and migration whereas siRNA-mediated knockdown of GPRC5A impaired proliferation and invasion. We found that gemcitabine treatment enhanced GPRC5A expression in pancreatic cancer cells. Conversely, siRNA-mediated knockdown of GPRC5A sensitized the cells to gemcitabine. We showed that HuR/ELAVL1 up-regulated GPRC5A protein in the first 18 hours following gemcitabine treatment by stabilizing GPRC5A’s mRNA through binding to at least on site in GPRC5A’s 3´UTR. Microarray analysis showed that GPRC5A overexpression impacts several signaling networks. Conclusions: Our results indicate that GPRC5A acts as an oncogene in pancreatic cancer. Unexpectedly, gemcitabine was found to increase GPRC5A's mRNA and protein levels. We showed that this increase is mediated by HuR, a known enabler of gemcitabine efficacy, through a direct interaction between HuR and GPRC5A’s mRNA. It appears that the sensitivity of pancreatic cancer cells to gemcitabine can be augmented through down-regulation of GPRC5A. Citation Format: Honglei Zhou, Aristeidis Telonis, Yi Jing, Masaya Jimbo, Fernando Blanco, Eric Londin, Jonathan Brody, Isidore Rigoutsos.{Authors}. GPRC5A acts as a potent oncogene in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A41.

ABC transporters are the active transport systems of the cell involved in the export or import of a wide variety of molecules. We discovered that a member of the ABC transporter family called ABCC3 has a critical role in pancreatic cancer. ABCC3 blockade using genetic knockdown inhibits pancreatic cancer growth in vitro and in vivo. In addition, we demonstrate that knockdown of ABCC3 reduces cell proliferation by inhibition of STAT3 and HIF1α signalling pathways, which are key regulators of pancreatic cancer progression. A focused chemical library of indenes was screened for ABCC3 inhibition using ABCC3 expressing pancreatic tumour cells. A drug development candidate, designated as S3, emerged following extensive chemical modification to optimize target selectivity and oral bioavailability. Oral administration of S3 significantly inhibited tumour growth and increased survival in several mouse models of pancreatic cancer without discernible toxicity. Interestingly, using the KPC transgenic mouse model that closely mimics human pancreatic cancer, we identified a dual activity of S3 to inhibit the growth of the primary tumour and impact the surrounding stroma. Strikingly, a significant increase in survival was achieved with S3 treatment compared to vehicle treated KPC mice. A two-fold increase in lifespan was observed from 72.5 days (median survival) in the control group to 146.5 days in the treatment group. Importantly, we observed no overt toxicity from S3 treatment at a dosage of 50 mg/kg, which generated plasma levels exceeding growth inhibitory IC50 values. Furthermore, we show that stromal cells in pancreatic tumours, which actively participate in cancer progression, are enriched for ABCC3, and that its inhibition may contribute to stroma reprogramming. In other studies, we found that S3 inhibits the closely related transporter, ABCC1, and that pharmacological inhibition of ABCC1 reduced prostate cancer cell growth in vitro and potentiated the effects of Docetaxel in vitro and in mouse models of prostate cancer in vivo. Mechanistically, we have shown that ABCC3, is overexpressed in pancreatic cancer cells and can efflux the bioactive lipid lysophosphatidylinositol (LPI) which, in turn, activates its receptor G protein-coupled receptor 55 in an autocrine mitogenic loop. Similarly, ABCC1 mediates LPI efflux in prostate cancer cells. The fact that both ABCC1 and ABCC3 transport LPI and are inhibited by S3 is not surprising considering that they share a high primary sequence identity and are known to have overlapping substrate specificity. Interestingly, unlike known ABC inhibitors, S3 has anticancer activity as a single agent. Our goal is to further study the antitumor activity of S3 alone and in combination with conventional chemotherapy or molecular targeted drugs used for the treatment of pancreatic and prostate cancer. Citation Format: Marco Falasca, Xi Chen, Gary Piazza. Novel ABC transport inhibitor as a treatment for pancreatic and prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1814.

Acinar-to-ductal metaplasia (ADM) is a precursor lesion of pancreatic ductal adenocarcinoma (PDAC); however, the regulators of the ADM-mediated PDAC development and its targeting are poorly understood. RNA polymerase II-associated factor 1 (PAF1) maintains cancer stem cells leading to the aggressiveness of PDAC. In this study, we investigated whether PAF1 is required for the YAP1-mediated PDAC development and whether CA3 and verteporfin, small molecule inhibitors of YAP1/TEAD transcriptional activity, diminish pancreatic cancer (PC) cell growth by targeting the PAF1/YAP1 axis. Here, we demonstrated that PAF1 co-expresses and interacts with YAP1 specifically in metaplastic ducts of mouse cerulein- or KrasG12D-induced ADM and human PDAC but not in the normal pancreas. PAF1 knockdown (KD) reduced SOX9 in PC cells, and the PC cells showed elevated PAF1/YAP1 complex recruitment to the promoter of SOX9. The PAF1 KD reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in the mouse KC (KrasG12D; Pdx-1 Cre) cells and human PC cells, indicating that the PAF1 is required for the YAP1-mediated development of ADM and PC. Moreover, treatment with CA3 or verteporfin reduced the expressions of PAF1, YAP1, TEAD4, and SOX9 and decreased colony formation and stemness in KC and PC cells. CA3 treatment also reduced the viability and proliferation of PC cells and diminished the duct-like structures in KC acinar explants. CA3 or verteporfin treatment decreased the recruitment of the PAF1/YAP1 complex to the SOX9 promoter in PC cells and reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in KC and PC cells. Overall, PAF1 cooperates with YAP1 during ADM and PC development, and verteporfin and CA3 inhibit ADM and PC cell growth by targeting the PAF1/YAP1/SOX9 axis in vitro and ex vivo models. This study identified a regulatory axis of PDAC initiation and its targeting, paving the way for developing targeted therapeutic strategies for pancreatic cancer patients.

The Hirshberg Foundation for Pancreatic Cancer Research was founded by Agi Hirshberg in 1997, following her husband Ronald Hirshberg’s 8-month, 7-day battle with the disease. (http://www.pancreatic.org/). At the time, the Hirshberg family viewed pancreatic cancer as the ‘‘unknown enemy’’ (http:// www.pancreatic.org/site/c.htJYJ8MPIwE/b.8276835/k.66CB/ Agi_Hirshbergs_Story.htm). In the years since Ronald Hirshberg’s death, Agi Hirshberg has made it her mission to unmask the unknown for others facing the same enemy. When patients present with a new diagnosis of pancreatic cancer, most are already in an advanced stage of the disease. Pancreatic cancer is resistant to most chemotherapeutic agents and radiotherapy. These factors combine to make pancreatic cancer a highly lethal disease, and according to the National Cancer Institute (NCI) 2013 estimates, pancreatic cancer was diagnosed in approximately 45,220 US citizens, and some 38,460 died of the disease. 1 The Annual Report to the Nation on the Status of Cancer, 1975Y2009, shows that overall cancer death rates continued to decline in the United States among both men and women, among all major racial and ethnic groups, and for all of the many common cancer sites, including lung, colon and rectum, female breast, and prostate. However, this trend does not hold true for pancreatic cancer. The report demonstrates that death rates continued to increase during the latest time period (2000 through 2009) for pancreatic cancer. 2 Only about 23% of patients with cancer of the pancreas will still be living 1 year after diagnosis, and only about 4% will survive 5 years. Even for those diagnosed with pancreatic cancer that has not spread to other organs or systems, the overall 5-year survival rate approaches only 15%. The Hirshberg Foundation’s vision is to advance pancreatic cancer research and provide information, resources, and support to pancreatic cancer patients and their families. The mission of the Foundation is as follows: & To find a cure for pancreatic cancer in honor of Ron Hirshberg and the thousands of people who are diagnosed with this disease each year. & To create a premier Pancreatic Cancer Center where all needs of pancreatic cancer patients can be met in one location with the most advanced treatment options. & To be recognized as a patient support reference source for

Chronic Pancreatitis and Pancreatic Cancer: Prediction and Mechanism

RAS-RAF-miR-296-3p signaling axis increases Rad18 expression to augment radioresistance in pancreatic and thyroid cancers

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in United States. Tremendous efforts have witnessed the failure of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), extends patient survival by only a few weeks. Numerous studies implicate the need for the drugs that can target multiple signaling events in pancreatic cancer. Recently, cucurbitacins, tetracyclic triterpenoid compund/molecules, belonging to a family of Cucurbitaceae have shown promising anti-cancer activities such as antiproliferation, cell cycle arrest, and apoptosis induction. Here, we report the anticancer activity of a novel analogue of cucurbitacin (Cuc D) that is able to target important signaling proteins involved in pancreatic cancer cell proliferation, invasion and metastasis. Methods: Herein, we investigated the use of Cuc D for the treatment of pancreatic cancer using a panel of pancreatic cancer cells. The effect of Cuc D on the growth of pancreatic cancer cells was determined by CellTiter 96® AQueous One Solution cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96h. The clonogenic potential of cancer cells was also studied using the colony formation assay. microRNA-21 expression levels were investigated through Real-time PCR. Immunoblotting techniques were performed to determine the effects of Cuc D at the molecular level. Results: Our results indicate the anticancer effects of Cuc D in pancreatic cancer cells. Cuc D induces concentration dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell lines at nano molar concentrations. It also inhibits colony formation and metastasis potential of pancreatic cancer cells in dose and time dependent manner. Importantly, Cuc D targets and inhibits proteins, MUC13, Bcl2 and survivin (inhibitor of apoptosis protein family member: IAP) that are involved in proliferation, metastasis and tumor progression as seen through Western blotting. Additionally, the treatment of Cuc D induces the phosphorylation of p53 (p-p53; ser-15) in pancreatic cancer cells. We also observed that Cuc D effectively inhibits miR-21 levels in pancreatic cancer, HPAF-II cells as seen through Real time PCR analysis. miR-21 is an oncogenic miRNA that is overexpressed in pancreatic cancer. Conclusion: Our findings demonstrate that Cuc D exerts multi-focal action in pancreatic cancer cells targeting multiple signaling events. Overall, this study suggests that Cuc D can be a potential and promising therapeutic modality for pancreatic cancer treatment. Citation Format: Mohammed Sikander, Mohd Saif Zaman, Neeraj Chauhan, Murali M. Yallapu, Sheema Khan, Fathi T. Halaweish, Bhavin Chauhan, Meena Jaggi, Subhash C. Chauhan. A multi-targeted approach for pancreatic cancer treatment by a novel cucurbitacin analogue. [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 2714. doi:10.1158/1538-7445.AM2014-2714

This paper reports on the green synthesis of heparin-reduced gold nanoparticles and their nanotopography as studied with atomic force microscopy. The study also evaluated the anticoagulant activity of the newly prepared gold nanoparticles. The heparin-reduced gold nanoparticles were homogeneous, showing characteristic surface plasmon resonance bands of approximately 523-527 nm, and their shapes were mostly spherical and amorphous. The average diameter of the nanoparticles measured from atomic force microscopic images was either 20.26 +/- 3.35 nm or 40.85 +/- 8.95 nm depending on the different precursor salts and heparin concentrations. Atomic force microscopic images revealed that the topography of the heparin polymer aggregated when deposited onto mica, resembling a chain of mountains. This characteristic nanotopography of the heparin disappeared after the synthesis of the gold nanoparticles was performed. Interestingly, prolonged prothrombin time, thrombin time, and activated partial thromboplastin time were observed in the heparin-reduced gold nanoparticles when compared to a control heparin, suggesting the enhancement of anticoagulant activity in heparin-reduced gold nanoparticles. Hence, the green synthesis of gold nanoparticles with heparin using a simple reaction step could be a viable procedure for enhancing heparin's anticoagulant activity.