Pancreatic Ductal Adenocarcinoma Cancer Cells Research Articles

State of the art and future directions of pancreatic ductal adenocarcinoma therapy.

Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second cause of cancer-related death in 2030. PDAC is the poorest prognostic tumor of the digestive tract, with 80% of patients having advanced disease at diagnosis and 5-year survival rate not exceeding 7%. Until 2010, gemcitabine was the only validated therapy for advanced PDAC with a modest improvement in median overall survival as compared to best supportive care (5-6 vs 3 months). Multiple phase II-III studies have used various combinations of gemcitabine with other cytotoxics or targeted agents, most in vain, in attempt to improve this outcome. Over the past few years, the landscape of PDAC management has undergone major and rapid changes with the approval of the FOLFIRINOX and gemcitabine plus nab-paclitaxel regimens in patients with metastatic disease. These two active combination chemotherapy options yield an improved median overall survival (11.1 vs 8.5 months, respectively) thus making longer survival a reasonably achievable goal. This breakthrough raises some new clinical questions about the management of PDAC. Moreover, better knowledge of the environmental and genetic events that underpin multistep carcinogenesis and of the microenvironment surrounding cancer cells in PDAC has open new perspectives and therapeutic opportunities. In this new dynamic context of deep transformation in basic research and clinical management aspects of the disease, we gathered updated preclinical and clinical data in a multifaceted review encompassing the lessons learned from the past, the yet unanswered questions, and the most promising research priorities to be addressed for the next 5 years.

Abstract A30: A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation

Abstract To identify the cellular components that participate in the regulation of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, we carried out a genome-wide RNAi depletion screen. We employed a rabbit monoclonal antibody specific for RPS6 [Ser235P/Ser236P] and high content microscopy to quantify rpS6 phosphorylation in the pancreatic ductal adenocarcinoma cancer cell line (PDAC) MiaPaCa-2. Applying a stringent selection, we retrieved over 600 genes wherein at least two RNAi gave significant reduction in S6 phosphorylation. This cohort is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. To identify mTORC1 regulators that did not require the TSC, we examined which of the positives identified in MiaPaCa-2 cells were also required for S6 phosphorylation in TSC1 null mouse embryo fibroblasts. Among the 541 such gene products examined in TSC1 null MEFs, RNAi pools directed against 79 were found to reduce S6 phosphorylation significantly in 2 or 3 replicates. Among these, GPCRs, other transmembrane proteins and several signaling proteins comprised 30%, elements concerned with DNA structure and transcription, RNA processing and translation together comprised 28%, and the remainder distributed among proteins concerned with membrane transport and traffic, protein traffic and modification, mitochondria, metabolism and the cytoskeleton. Surprisingly, the only elements among this cohort previously associated with the maintenance of mTORC1 activity are two of the numerous subunits of the vacuolar ATPase subunits and the CUL4 subunit DDB1. RNAis against another 84 TSC1null MEF targets were observed to reduce S6 phosphorylation in only one of three replicates, however an indication that this group also bears attention is the presence of RPS6KB1 itself, as well as Rac1 and MAP4K3, a protein kinase that supports amino acid signaling of mTORC1 to RPS6KB1. Our screen confirmed previously known and proposed regulators of S6 phosphorylation and mTORC1 in addition to previously unidentified mTORC1 related proteins. Our results suggest the potential operation of feedback pathways from mRNA translation, RNA processing and DNA repair in the regulation of mTORC1. Since many cancers exhibit mTOR hyperactivation, identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new drug targets and lead to the development of beneficial therapies for tumors sensitive to mTOR inhibition. In addition, we found 43 genes that matched the mTOR essentiality profile in 216 different cancer cell lines represented in the Achilles dataset. These genes may help delineate a more general cellular state characterized by co-dependency of mTOR dependency and the respective P-S6 positive genes therefore providing the rationale for selecting specific proteins for further analysis as candidate drug targets. Citation Format: Angela Papageorgiou, Pablo Tamayo, Jill Mesirov, Joseph Avruch, Joseph Rapley. A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A30.

DUSP1 Is a Novel Target for Enhancing Pancreatic Cancer Cell Sensitivity to Gemcitabine

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by excessive mitogenic pathway activation and marked chemoresistance to a broad spectrum of chemotherapeutic drugs. Dual specificity protein phosphatase 1 (DUSP1) is a key negative regulator of mitogen activated protein kinases (MAPKs). Yet, DUSP1 is overexpressed in pancreatic cancer cells (PCCs) in PDAC where it paradoxically enhances colony formation in soft agar and promotes in vivo tumorigenicity. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human PCCs, we show that gemcitabine activates c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Gemcitabine-induced JNK and p38 MAPK activation mediates increased apoptosis, but also transcriptionally upregulates DUSP1, as evidenced by increased DUSP1 mRNA levels and RNA polymerase II loading at DUSP1 gene body. Conversely, shRNA-mediated inhibition of DUSP1 enhances JNK and p38 MAPK activation and gemcitabine chemosensitivity. Using doxycycline-inducible knockdown of DUSP1 in established orthotopic pancreatic tumors, we found that combining gemcitabine with DUSP1 inhibition improves animal survival, attenuates angiogenesis, and enhances apoptotic cell death, as compared with gemcitabine alone. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the advantage of enhancing gemcitabine chemosensitivity while suppressing angiogenesis.

Effect of extracellular matrix components on the expression of epithelial-to-mesenchymal transition markers in cultured human pancreatic ductal adenocarcinoma cells

Epithelial-to-mesenchymal transition (EMT) is a step-wise process leading to the phenotypic switch of epithelial to mesenchymal cells, providing these cells with a metastatic phenotype. During EMT epithelial cells loose adhesion by down regulation of E-cadherin and express N-cadherin, display cytoskeleton reorganization by expressing vimentin and α-smooth muscle actin (αSMA), acquire motile properties and become invasive by secretion of matrix metalloproteinases (MMPs). Cancer cell phenotype is influenced by the tumor microenvironment in relation to tumor progression, as well as to cell proliferation and invasion. The role of the extracellular matrix (ECM) in the microenvironment is particularly relevant in pancreatic ductal adenocarcinoma (PDAC) since this carcinoma is characterized by an intense desmoplastic reaction, representing the environment where the complex interplay between tumor cells, stromal fibroblasts and ECM components occurs. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and their microenvironment by characterizing PDAC cell phenotype in cells cultured on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell behaviour and the proteins occurring in the desmoplastic tissue. We analyzed by immunofluorescence the expression of the main EMT markers such as E-cadherin, N-cadherin, β-catenin, αSMA, vimentin and collagen type I (COL-I) in PDAC cells cultured on laminin, fibronectin, COL-I and without coating (NC). Moreover, we investigated cell proliferation and MMPs activity in cell culture supernatants by SDS-zymography. Cell morphology was similar in PDAC cells cultured on laminin, fibronectin, COL-I, and in NC, as well as the E-cadherin/β-catenin complex, αSMA and COLI expression; by contrast, vimentin was undetectable in all the experimental conditions. N-cadherin was slightly detectable in cells cultured on fibronectin, COL-I, and laminin, and at lower extent in NC cells. Cell proliferation resulted similar in NC and in cells cultured on fibronectin, decreased on laminin and increased on COL-I. MMP-9 activity exhibited a similar trend, resulting similar on fibronectin, decreased on laminin and stimulated on COL-I. These preliminary results provide new insights in the characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell, and will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies.

Mechanistic Insights into Self-Reinforcing Processes Driving Abnormal Histogenesis During the Development of Pancreatic Cancer

Pancreatic ductal adenocarcinoma, one of the most feared lethal and painful diseases, is increasing in incidence. The poor prognosis of pancreatic ductal adenocarcinoma-affected patients primarily is owing to our inability to develop effective therapies. Mechanistic studies of genetic, epigenetic, and cell-to-cell signaling events are providing clues to molecular pathways that can be targeted in an attempt to cure this disease. The current review article seeks to draw inferences from available mechanistic knowledge to build a theoretical framework that can facilitate these approaches. This conceptual model considers pancreatic cancer as a tissue disease rather than an isolated epithelial cell problem, which develops and progresses in large part as a result of three positive feedback loops: i) genetic and epigenetic changes in epithelial cells modulate their interaction with mesenchymal cells to generate a dynamically changing process of abnormal histogenesis, which drives more changes; ii) the faulty tissue architecture of neoplastic lesions results in unsynchronized secretion of signaling molecules by cells, which generates an environment that is poor in oxygen and nutrients; and iii) the increased metabolic needs of rapidly dividing cells serve as an evolutionary pressure for them to adapt to this adverse microenvironment, leading to the emergence of resistant clones. We discuss how these concepts can guide mechanistic studies, as well as aid in the design of novel experimental therapeutics.

S100P is a metastasis-associated gene that facilitates transendothelial migration of pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is the 5th most common cause of cancer death in the UK and the 4th in the US. The vast majority of deaths following pancreatic cancer are due to metastatic spread, hence understanding the metastatic process is vital for identification of critically needed novel therapeutic targets. An enriched set of 33 genes differentially expressed in common between primary PDAC and liver metastases, when compared to normal tissues, was obtained through global gene expression profiling. This metastasis-associated gene set comprises transcripts from both cancer (S100P, S100A6, AGR2, etc.) and adjacent stroma (collagens type I, III, and V, etc.), thus reinforcing the concept of a continuous crosstalk between the two compartments in both primary tumours and their metastases. The expression of S100P, SFN, VCAN and collagens was further validated in additional primary PDACs and matched liver metastatic lesions, while the functional significance of one of the most highly expressed genes, S100P, was studied in more detail. We show that this protein increases the transendothelial migration of PDAC cancer cells in vitro, which was also confirmed in vivo experiments using a zebrafish embryo model. Thus S100P facilitates cancer cell intravasation/extravasation, critical steps in the hematogenous dissemination of pancreatic cancer cells.

A feasibility study on gene therapy of pancreatic carcinoma with Ad-PUMA

Pancreatic cancer is one of the most malignant tumors with high mortality and poor prognosis even with the aggressive conventional therapies. Biotherapy based on the understanding of tumorigenesis mechanism is ongoing to improve the outcomes of cancer patients. We sought here to evaluate the therapeutic potential of a proapoptotic gene, PUMA, in pancreatic cancer. We found that PUMA was differently expressed in a series of pancreatic ductal adenocarcinoma cancer cell lines, and adenovirus-mediated expression of PUMA (Ad-PUMA) in these cells resulted in massive apoptosis. PUMA was more potent than p53 in suppressing growth of cancer cells. RT-PCR and Western Blot revealed that exogenous PUMA was expressed 6 h after Ad-PUMA infection. Furthermore, we assessed the efficacy of Ad-PUMA combining anticancer drugs (5-fluorouracil, cisplatin, gemcitabine hydrochloride, respectively) in these pancreatic cancer cell lines. Data revealed that PUMA significantly sensitized pancreatic carcinoma cell lines to chemotherapeutics, which may be resulted from abundant apoptosis induction. In nude mice with PANC-1 xenografts, Ad-PUMA treatment significantly inhibited the tumor growth. These results suggest that PUMA is a potent molecular tool in suppressing tumor growth sensitizing pancreatic carcinoma cells to chemical drugs. PUMA plays roles in negatively regulating cancer cell growth and may be a promising tool for cancer biotherapy, with or without combination with chemotherapeutic agents.

Network Modeling of MDM2 Inhibitor-Oxaliplatin Combination Reveals Biological Synergy in wt-p53 solid tumors

Earlier we had shown that the MDM2 inhibitor (MI-219) belonging to the spiro-oxindole family can synergistically enhance the efficacy of platinum chemotherapeutics leading to 50% tumor free survival in a genetically complex pancreatic ductaladenocarcinoma (PDAC) xenograft model. In this report, we have taken a systems and network modeling approach in order to understand central mechanisms behind MI219-oxaliplatin synergy with validation in PDAC, colon and breast cancer cell lines. Microarray profiling of drug treatments (MI-219, oxaliplatin or their combination) in capan-2 cells reveal a similar unique set of gene alterations that is duplicated in other solid tumor cells. As single agent, MI-219 or oxaliplatin induced alterations in 48 and 761 genes respectively. The combination treatment resulted in 767 gene alterations with emergence of 286 synergy unique genes. Ingenuity network modeling of combination and synergy unique genes showed the crucial role of five key local networks CREB, CARF, EGR1, NF-kB and E-Cadherin. Compared to single agents the combination treatment super induced p53 and p21 confirming functional synergy. Further, the network signatures were validated at the protein level in all three cell lines. Individually silencing central nodes in these five hubsinterfered with MI-219-oxaliplatin activity confirming their critical role in aiding p53 mediated apoptotic response. We anticipate that our MI219-oxaliplatin network blueprints can be clinically translated in the rationale design and application of this unique therapeutic combination in a genetically pre-defined subset of patients.

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Ras-mediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.

GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the deregulation of the hedgehog signaling pathway. The Sonic Hedgehog ligand (Shh), absent in the normal pancreas, is highly expressed in pancreatic tumors and is sufficient to induce neoplastic precursor lesions in mouse models. We investigated the mechanism of Shh signaling in PDAC carcinogenesis by genetically ablating the canonical bottleneck of hedgehog signaling, the transmembrane protein Smoothened (Smo), in the pancreatic epithelium of PDAC-susceptible mice. We report that multistage development of PDAC tumors is not affected by the deletion of Smo in the pancreas, demonstrating that autocrine Shh-Ptch-Smo signaling is not required in pancreatic ductal cells for PDAC progression. However, the expression of Gli target genes is maintained in Smo-negative ducts, implicating alternative means of regulating Gli transcription in the neoplastic ductal epithelium. In PDAC tumor cells, we find that Gli transcription is decoupled from upstream Shh-Ptch-Smo signaling and is regulated by TGF-beta and KRAS, and we show that Gli1 is required both for survival and for the KRAS-mediated transformed phenotype of cultured PDAC cancer cells.

Targeting angiogenesis in pancreatic cancer: rationale and pitfalls

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer responsible for over 20% of deaths due to gastrointestinal malignancies. PDAC is usually diagnosed at an advanced stage which, in part, helps to explain its high resistance to chemotherapy and radiotherapy. In addition, the cancer cells in PDAC have a high propensity to metastasize and to aberrantly express several key regulators of angiogenesis and invasion. Chemotherapy has only provided a modest impact on mean survival and often induces side effects. Targeting angiogenesis alone or in combination with other modalities should be investigated to determine if it may provide for increased survival. This review summarizes the alterations in PDAC that play a critical role in angiogenesis and provides an overview of current and therapeutic strategies that may be useful for targeting angiogenesis in this malignancy.

Aberrant expression of neuropilin-1 and -2 in human pancreatic cancer cells.

Neuropilin (Np)-1 and -2 are coreceptors for vascular endothelial growth factor (VEGF). This study was designed to assess their role in pancreatic ductal adenocarcinoma (PDAC). We assessed Np-1 and Np-2 expression by real-time quantitative PCR in relation to the expression of VEGF ligands and receptors in pancreatic cancer cell lines and tissues. ASPC-1, CAPAN-1, and PANC-1 pancreatic cancer cells and tumor-derived, laser-captured pancreatic cancer cells exhibited higher Np-1 and Np-2 mRNA levels than VEGF receptor-1, -2, or -3 mRNA levels. Transfection of Np-1 and Np-2 cDNAs in COS-7 cells, and treatment with tunicamycin revealed that both proteins were glycosylated. Both proteins were expressed in pancreatic cancer cell lines, in the PDAC samples, and in acinar cells adjacent to the cancer cells. The normal pancreas was devoid of Np-1 immunoreactivity, whereas Np-2 immunoreactivity was present in the endocrine islets and in some acinar cells, but not in ductal cells. The aberrant localization of Np-1 and Np-2 in the cancer cells in PDAC suggests that in addition to exerting proangiogenic effects, these coreceptors may contribute to novel autocrine-paracrine interactions in this malignancy.

Epiregulin Is Up-Regulated in Pancreatic Cancer and Stimulates Pancreatic Cancer Cell Growth

Epiregulin belongs to the epidermal growth factor (EGF) family of polypeptides. Previous studies have underscored the important role of the EGF family of ligands and receptors in the pathology of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). It is not known, however, whether epiregulin may also have a role in these diseases. Therefore, in the present study we investigated the expression and function of epiregulin in five pancreatic cancer cell lines and in PDAC and CP tissue samples. Epiregulin mRNA was present at high (MIA-PaCa-2 cells) or moderate levels (ASPC-1, CAPAN-1, and T3M4) in most cells, but was below detection levels in PANC-1 cells. All the cell lines exhibited a dose-dependent increase in growth in response to recombinant human epiregulin. Epiregulin mRNA levels were increased 2.1-fold in PDAC samples (P < 0.01) and 1.7-fold in CP samples (P < 0.01), when compared with the normal controls. There was no correlation between epiregulin mRNA levels and tumor stage or grade. By in situ hybridization, a moderate to intense epiregulin mRNA signal was present in most pancreatic cancer cells in PDAC. In contrast, only a weak (normal pancreas) to moderate (CP) signals were present in the ductal and acinar cells in CP. These findings suggest that epiregulin may contribute to the pathobiology of PDAC, and may also have a role in CP.