Induction of invasive mouse skin carcinomas in transgenic mice with mutations in both H-ras and p53

Synergistic interaction between H-ras and p53 were systematically examined during skin tumorigenesis. Concurrent expression of an activated H-ras gene and a mutant p53 gene was accomplished by crossing p53(Val135/wt) mice with TG.AC mice. Topical application to wild-type mice with benzo(a)pyrene (BaP) alone produced approximately 26% skin tumor incidence, whereas BaP treatment of p53(wt/wt)Hras(TG.AC/wt), p53(Val135/wt)Hras(wt/wt), and p53(Val135/wt)Hras(TG.AC/wt) mice produced a 75%, 77%, and 100% incidence of skin tumors, respectively. An average of 0.33 tumor per mouse was observed in wild-type (p53(wt/wt)Hras(wt/wt)) mice, whereas approximately 1.54, 1.96, and 3.08 tumors per mouse were seen in BaP-treated p53(wt/wt)Hras(TG.AC/wt), p53(Val135/wt)Hras(wt/wt), and p53(Val135/wt)Hras(TG.AC/wt) mice, respectively. The effects on total tumor volume were even more striking with 7-, 48-, and 588-fold increases in tumor volume compared with wild-type (p53(wt/wt)Hras(wt/wt)) in p53(wt/wt)Hras(TG.AC/wt), p53(Val135/wt)Hras(wt/wt), and p53(Val135/wt)Hras(TG.AC/wt) mice, respectively. Histopathologically, all tumors from p53(wt/wt)Hras(wt/wt) mice were either papillomas or well-differentiated squamous cell carcinomas, whereas the tumors in p53(wt/wt)Hras(TG.AC/wt), p53(Val135/wt)Hras(wt/wt), and p53(Val135/wt)Hras(TG.AC/wt) mice were principally squamous cell carcinomas with varying degree of invasiveness. Particularly, tumors in p53(Val135/wt)Hras(TG.AC/wt) mice exhibited the most rapid growth and the extreme form of tumor invasion. Microarray analysis revealed that dominant-negative p53 (Val135) and activated H-ras affected several cellular processes involved in tumorigenesis possibly through its effects on apoptosis, cell cycle arrest, and Ras-mitogen-activated protein kinase pathways. The present study provides the first in vivo evidence that a germ line p53 mutation and activated H-ras act synergistically to profoundly enhance tumor progression.     

Autoantibody signature in human ductal pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. It is the fourth leading cause of cancer death with a 2% 5-year survival rate. Biomarkers for its early detection are lacking. This study was designed to use a proteomics-based approach as a means of identifying antigens that elicit a humoral response in PDAC patients. Antibodies against PDAC-associated antigens are useful for early cancer diagnosis and therapy. Proteins from PDAC cell lines were separated by 2-DE, and the serum IgG reactivity of 70 PDAC patients, 40 healthy subjects (HS), 30 non-PDAC tumor patients, and 15 chronic pancreatitis (CP) patients was tested by Western blot analysis. Spots specifically recognized by PDAC sera and revealed by mass spectrometry corresponded to metabolic enzymes or cytoskeletal proteins. Most were up-regulated in PDAC tissues. Thus, it seems that metabolic enzymes and cytoskeletal proteins are specific targets of the humoral response during PDAC. The results of further studies of these serological-defined antigens could be of diagnostic and therapeutic significance in PDAC.     

Proteogenomic characterization of pancreatic ductal adenocarcinoma

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Microvascular density in chronic pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic adenocarcinoma represent two pathologic phenomena with marked production of connective tissue stroma containing numerous small blood vessels. The aim of this study was to characterise quantitatively the vascular supply of pancreatic adenocarcinoma and fragments of the periductal tissue collected from patients with chronic pancreatitis. The study material included 18 cases of pancreatitis and 22 cases of pancreatic ductal adenocarcinoma. Microvessels were marked using monoclonal anti-CD34 antibodies. The number of blood vessels in the fibrous stroma was significantly higher in the chronic pancreatitis samples compared to the pancreatic carcinoma group (mean vessel count 298 and 194 vessel/mm2; median 251 and 187 vessel/mm2 respectively; p<0.01). Distributions of the vascular diameter in both studied groups were very similar. The obtained results suggest that the development of vascular network accompanying chronic pancreatitis is more effective in some parts of pancreas compared to angiogenic intensity in pancreatic adenocarcinoma.     

The ARF tumor suppressor inhibits tumor cell colonization independent of p53 in a novel mouse model of pancreatic ductal adenocarcinoma metastasis

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.     

Stroma and pancreatic ductal adenocarcinoma: An interaction loop

Pancreatic ductal adenocarcinom a (PDA) has two exceptional features. First, it is a highly lethal disease, with a median survival of less than 6months and a 5-year survival rate less than 5%. Second, PDA tumor cells are surrounded by an extensive stroma, which accounts for up to 90% of the tumor volume. It is well recognized that stromal microenvironment can accelerate malignant transformation, tumor growth and progression. More importantly, the interaction loop between PDA and its stroma greatly contributes to tumor growth and progression. We propose that the extensive stroma of PDA is closely linked to its poor prognosis. An improved understanding of the mechanisms that contribute to pancreatic tumor growth and progression is therefore urgently needed. Targeting the stroma may thus provide novel prevention, earlier detection and therapeutic options to this deadly malignancy. Accordingly, in this review, we will summarize the mechanism of PDA stroma formation, the role of the stroma in tumor progression and therapy resistance and the potential of stroma-targeted therapeutics strategies.     

Targeted nanoparticles for imaging incipient pancreatic ductal adenocarcinoma

Background

Pancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, typically presenting with metastasis at the time of diagnosis and exhibiting profound resistance to existing therapies. The development of molecular markers and imaging probes for incipient PDAC would enable earlier detection and guide the development of interventive therapies. Here we sought to identify novel molecular markers and to test their potential as targeted imaging agents.

Methods and Findings

Here, a phage display approach was used in a mouse model of PDAC to screen for peptides that specifically bind to cell surface antigens on PDAC cells. These screens yielded a motif that distinguishes PDAC cells from normal pancreatic duct cells in vitro, which, upon proteomics analysis, identified plectin-1 as a novel biomarker of PDAC. To assess their utility for in vivo imaging, the plectin-1 targeted peptides (PTP) were conjugated to magnetofluorescent nanoparticles. In conjunction with intravital confocal microscopy and MRI, these nanoparticles enabled detection of small PDAC and precursor lesions in engineered mouse models.

Conclusions

Our approach exploited a well-defined model of PDAC, enabling rapid identification and validation of PTP. The developed specific imaging probe, along with the discovery of plectin-1 as a novel biomarker, may have clinical utility in the diagnosis and management of PDAC in humans.     

Bracken fern-induced malignant tumors in rats: absence of mutations in p53, H-ras and K-ras and no microsatellite instability

Bracken fern (genus Pteridium) has been shown to induce tumors in domestic and experimental animals. Epidemiological studies have also shown an association between human exposure to bracken toxins and increased risk for the development of upper gastrointestinal tract tumors. Our aim in this study was to investigate possible genomic alterations in bracken fern-induced tumors of experimental animals searching for molecular markers that might be used for human epidemiological studies. Using human colorectal carcinogenesis as a molecular model, we examined eight malignant bracken fern-induced tumors of rats for mutations in the genes associated with the "classic pathway" of colorectal cancer, i.e. p53 and ras, and also in the "mutator pathway" by evaluating microsatellite instability. Exons 5-9 of the p53 gene and exons 1 and 2 of the K-ras and H-ras genes were examined by DNA sequencing and no mutations were found in any of the eight tumors. Amplification of five previously validated microsatellite loci (one with mono-, three with di- and one with tetra-nucleotide repeat motifs) in the malignant tumors and in the surrounding normal tissue did not reveal any instability. The involvement of epigenetic alterations or of mutations in other tumor suppressor genes or oncogenes should be further investigated in the search for human epidemiological markers.     

Fibroblast heterogeneity in pancreatic ductal adenocarcinoma: Perspectives in immunotherapy

Cancer-associated fibroblasts (CAFs), the key component in pancreatic tumor microenvironment (TME), originate from many sources and are naturally heterogeneous in phenotype and function. Numerous studies have identified their crucial role in promoting tumorigenesis through many routes including fostering cancer proliferation, angiogenesis, invasion, and metastasis. Conversely, research also indicates that subsets of CAFs express anti-tumor activity. These dual effects reflect the complexity of CAF heterogeneity and their interactions with other cells and factors in pancreatic TME. A critical component in this environment is infiltrated immune cells and immune mediators, which can communicate with CAFs. The crosstalk occurs via the production of various cytokines, chemokines, and other mediators and shapes the immunological state in TME. Comprehensive studies of the crosstalk between CAFs and tumor immune environment, particularly internal mechanisms interlinking CAFs and immune effectors, may provide new approaches for pancreatic ductal adenocarcinoma (PDAC) treatments. In this review, we explore the characteristics of CAFs, describe the interplay among CAFs, infiltrated immune cells, other mediators, and provide an overview of recent CAF-target therapies, their limitations, and potential research directions in CAF in the context of PDAC.     

Acinar to ductal cell trans-differentiation: A prelude to dysplasia and pancreatic ductal adenocarcinoma

Pancreatic cancer (PC) is the deadliest neoplastic epithelial malignancies and is projected to be the second leading cause of cancer-related mortality by 2024. Five years overall survival being ~10%, mortality and incidence rates are disturbing. Acinar to ductal cell metaplasia (ADM) encompasses cellular reprogramming and phenotypic switch-over, making it a cardinal event in tumor initiation. Differential cues and varied regulatory factors drive synchronous functions of metaplastic cell populations leading to multiple cell fates and physiological outcomes. ADM is a precursor for developing early pre-neoplastic lesions further progressing into PC due to oncogenic signaling. Hence delineating molecular events guiding tumor initiation may provide cues for regenerative medicine and precision onco-medicine. Therefore, understanding PC pathogenesis and early diagnosis are crucial. We hereby provide a timely overview of the current progress in this direction and future perspectives we foresee unfolding in the best interest of patient well-being and better clinical management of PC.     

Stem cells as the root of pancreatic ductal adenocarcinoma

Emerging evidence suggests that stem cells play a crucial role not only in the generation and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.     

Differential expression and bioinformatics analysis of exosome circRNAs in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a fatal malignant tumor with an unfavorable prognosis. Increasing evidence indicated circRNAs were associated with the pathogenesis and progression of tumors, but data on the expression of serum exosomal circRNAs in PDAC are scarce. This study attempted to explore the prognostic value and function of serum exosomes in PDAC patients.MethodsMicroarray-based circRNA expression was determined in PDAC and paired with normal serum samples, and the intersection of differentially expressed circRNAs (DECs) in serum exosomal samples and GSE79634 tissue samples was conducted. A specific CircRNA database was applied to investigate DECs binding miRNAs. Target genes were predicted using the R package multiMiR. Cox regression analyses were applied for constructing a prognostic model. The immunological characteristics analysis was carried out through the TIMER, QUANTISEQ, XCELL, EPIC, and ssGSEA algorithms.Results15 DECs were finally identified, and a circRNA-miRNA-mRNA network was established. A prognostic risk model was developed to categorize patients according to the risk scores. Furthermore, the association between risk score and immune checkpoint genes including CD80, TNFSF9, CD276, CD274, LGALS9, and CD44 were significantly elevated in the high-risk group, while ICOSLG and ADORA2A were upregulated in the low-risk group.ConclusionsOur results may provide new clues for the prognosis and treatment of PDAC.     

HDAC2 promotes loss of primary cilia in pancreatic ductal adenocarcinoma

Loss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells. Inactivation of HDAC2 results in decreased Aurora A expression, which promotes disassembly of primary cilia. We further showed that HDAC2 controls ciliogenesis independently of Kras, which facilitates Aurora A expression. These studies suggest that HDAC2 is a novel regulator of primary cilium formation in PDAC cells.     

Amplified centrosomes may underlie aggressive disease course in pancreatic ductal adenocarcinoma

Centrosome amplification (CA), the presence of centrosomes that are abnormally numerous or enlarged, is a well-established driver of tumor initiation and progression associated with poor prognosis across a diversity of malignancies. Pancreatic ductal adenocarcinoma (PDAC) carries one of the most dismal prognoses of all cancer types. A majority of these tumors are characterized by numerical and structural centrosomal aberrations, but it is unknown how CA contributes to the disease and patient outcomes. In this study, we sought to determine whether CA was associated with worse clinical outcomes, poor prognostic indicators, markers of epithelial-mesenchymal transition (EMT), and ethnicity in PDAC. We also evaluated whether CA could precipitate more aggressive phenotypes in a panel of cultured PDAC cell lines. Using publicly available microarray data, we found that increased expression of genes whose dysregulation promotes CA was associated with worse overall survival and increased EMT marker expression in PDAC. Quantitative analysis of centrosomal profiles in PDAC cell lines and tissue sections uncovered varying levels of CA, and the expression of CA markers was associated with the expression of EMT markers. We induced CA in PDAC cells and found that CA empowered them with enhanced invasive and migratory capabilities. In addition, we discovered that PDACs from African American (AA) patients exhibited a greater extent of both numerical and structural CA than PDACs from European American (EA) patients. Taken together, these findings suggest that CA may fuel a more aggressive disease course in PDAC patients.     

Tyrosine nitration of c-SRC tyrosine kinase in human pancreatic ductal adenocarcinoma

During pancreatic tumorigenesis, the equilibrium between cell survival and cell death is altered, allowing aggressive neoplasia and resistance to radiation and chemotherapy. Local oxidative stress is one mechanism regulating programmed cell death and growth and may contribute to both tumor progression and suppression. Our recent in situ immunohistochemical studies demonstrated that levels of total nitrotyrosine, a footprint of the reactive nitrogen species peroxynitrite, are elevated in human pancreatic ductal adenocarcinomas. In this study, quantitative HPLC-EC techniques demonstrated a 21- to 97-fold increase in the overall levels of nitrotyrosine of human pancreatic tumor extracts compared to normal pancreatic extracts. Western blot analysis of human pancreatic tumor extracts showed that tyrosine nitration was restricted to a few specific proteins. Immunoprecipitation coupled with Western analysis identified c-Src tyrosine kinase as a target of both tyrosine nitration and tyrosine phosphorylation. Peroxynitrite treatment of human pancreatic carcinoma cells in vitro resulted in increased tyrosine nitration and tyrosine phosphorylation of c-Src kinase, increased (>2-fold) c-Src kinase activity, and increased association between c-Src kinase and its downstream substrate cortactin. Collectively, these observations suggest that peroxynitrite-mediated tyrosine nitration and tyrosine phosphorylation of c-Src kinase may lead to enhanced tyrosine kinase signaling observed during pancreatic ductal adenocarcinoma growth and metastasis.