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Pancreatic ductal adenocarcinoma is a devastating disease, characterized by a rapid progression and poor treatment response. Using gene expression profiling of pancreatic cancer tissues, we previously identified periostin as a potential diagnostic and therapeutic target. In this study, we report the overexpression of periostin in a larger set of pancreatic cancer tissues and show that although the periostin transcript is exclusively expressed in tumour cells, the protein product is only detected in the extracellular matrix adjacent to cancer cells. Using an enzyme-linked immunosorbent assay (ELISA) assay, we show significantly increased levels of periostin in the sera of pancreatic cancer patients compared to non-cancer controls. We demonstrate that periostin promotes the invasiveness of tumour cells by increasing the motility of cells without inducing expression of proteases, and enhances the survival of tumour cells exposed to hypoxic conditions. At the molecular level, we provide evidence that the α6β4 integrin complex acts as the cell receptor of periostin in pancreatic cancer cells and that interaction promotes phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT) though activation of the PI3 kinase pathway, but not the RAS/MEK/ERK pathway. These findings suggest an important role of periostin in pancreatic cancer and provide a rationale to study periostin for diagnostic and therapeutic applications.Oncogene (2007) 26, 2082–2094. doi:10.1038/sj.onc.1210009; published online 9 October 2006 [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is highly heterogeneous. Our understanding of full molecular and immune landscape of ESCC remains limited, hindering the development of personalised therapeutic strategies. To address this, we perform genomic-transcriptomic characterizations and AI-aided histopathological image analysis of 120 Chinese ESCC patients. Here we show that ESCC can be categorized into differentiated, metabolic, immunogenic and stemness subtypes based on bulk and single-cell RNA-seq, each exhibiting specific molecular and histopathological features based on an amalgamated deep-learning model. The stemness subgroup with signature genes, such as WFDC2, SFRP1, LGR6 and VWA2, has the poorest prognosis and is associated with downregulated immune activities, a high frequency of EP300 mutation/activation, functional mutation enrichment in Wnt signalling and the highest level of intratumoural heterogeneity. The immune profiling by transcriptomics and immunohistochemistry reveals ESCC cells overexpress natural killer cell markers XCL1 and CD160 as immune evasion. Strikingly, XCL1 expression also affects the sensitivity of ESCC cells to common chemotherapy drugs. This study opens avenues for ESCC treatment and provides a valuable public resource to better understand ESCC., (© 2024. The Author(s).)

Precise vascular patterning is crucial for normal growth and development. The ERG transcription factor drives Delta-like ligand 4 (DLL4)/Notch signalling and is thought to act as a pivotal regulator of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC-specific focal adhesion kinase (FAK)-knockout (KO) and point-mutant FAK-knock-in mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid immunoprecipitation mass spectrometry of endogenous proteins identified that endothelial nuclear-FAK interacts with the deubiquitinase USP9x and the ubiquitin ligase TRIM25. Further in silico analysis confirms that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel-sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x., Competing Interests: Competing interests K.M.H-D. is a scientific advisor for Ellipses Pharma and is a joint applicant on patent claims N421127GB and N417173GB. All other authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

MHC class II expression is a hallmark of professional antigen-presenting cells and key to the induction of CD4+ T helper cells. We found that these molecules are ectopically expressed on tumor cells in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC) and on several PDAC cell lines. In contrast to the previous reports that tumoral expression of MHC-II in melanoma enabled tumor cells to evade immunosurveillance, the expression of MHC-II on PDAC cells neither protected cancer cells from Fas-mediated cell death nor caused T-cell suppression by engagement with its ligand LAG-3 on activated T-cells. In fact and surprisingly, the MHC-II/LAG-3 pathway contributed to CD4+ and CD8+ T-cell cytotoxicity toward MHC-II-positive PDAC cells. By combining bioinformatic tools and cell-based assays, we identified a number of immunogenic neo-antigens that can be presented by the patients' HLA class II alleles. Furthermore, CD4+ T-cells stimulated with neo-antigens were capable of recognizing and killing a human PDAC cell line expressing the mutated genes. To expand this approach to a larger number of PDAC patients, we show that co-treatment with IFN-γ and/or MEK/HDAC inhibitors induced tumoral MHC-II expression on MHC-II-negative tumors that are IFN-γ-resistant. Taken together, our data point to the possibility of harnessing MHC-II expression on PDAC cells for neo-antigen-based immunotherapy., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 Queen Mary University of London. Published with license by Taylor & Francis Group, LLC.)

Background: Pancreatic cancer remains one of the most lethal cancers and is refractory to immunotherapeutic interventions. Oncolytic viruses are a promising new treatment option, but current platforms demonstrate limited efficacy, especially for inaccessible and metastatic cancers that require systemically deliverable therapies. We recently described an oncolytic vaccinia virus (VV), VVLΔTKΔN1L, which has potent antitumor activity, and a regime to enhance intravenous delivery of VV by pharmacological inhibition of pharmacological inhibition of PI3 Kinase δ (PI3Kδ) to prevent virus uptake by macrophages. While these platforms improve the clinical prospects of VV, antitumor efficacy must be improved., Methods: VVLΔTKΔN1L was modified to improve viral spread within and between tumors via viral B5R protein modification, which enhanced production of the extracellular enveloped virus form of VV. Antitumor immunity evoked by viral treatment was improved by arming the virus with interleukin-21, creating VVL-21. Efficacy, functional activity and synergy with α-programmed cell death protein 1 (α-PD1) were assessed after systemic delivery to murine and Syrian hamster models of pancreatic cancer., Results: VVL-21 could reach tumors after systemic delivery and demonstrated antitumor efficacy in subcutaneous, orthotopic and disseminated models of pancreatic cancer. The incorporation of modified B5R improved intratumoural accumulation of VV. VVL-21 treatment increased the numbers of effector CD8+ T cells within the tumor, increased circulating natural killer cells and was able to polarize macrophages to an M1 phenotype in vivo and in vitro. Importantly, treatment with VVL-21 sensitized tumors to the immune checkpoint inhibitor α-PD1., Conclusions: Intravenously administered VVL-21 successfully remodeled the suppressive tumor-microenvironment to promote antitumor immune responses and improve long-term survival in animal models of pancreatic cancer. Importantly, treatment with VVL-21 sensitized tumors to the immune checkpoint inhibitor α-PD1. Combination of PI3Kδ inhibition, VVL-21 and α-PD1 creates an effective platform for treatment of pancreatic cancer., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Tumor-targeting oncolytic viruses such as vaccinia virus (VV) are attractive cancer therapeutic agents that act through multiple mechanisms to provoke both tumor lysis and anti-tumor immune responses. However, delivery of these agents remains restricted to intra-tumoral administration, which prevents effective targeting of inaccessible and disseminated tumor cells. In the present study we have identified transient pharmacological inhibition of the leukocyte-enriched phosphoinositide 3-kinase δ (PI3Kδ) as a novel mechanism to potentiate intravenous delivery of oncolytic VV to tumors. Pre-treatment of immunocompetent mice with the PI3Kδ-selective inhibitor IC87114 or the clinically approved idelalisib (CAL-101), prior to intravenous delivery of a tumor-tropic VV, dramatically improved viral delivery to tumors. This occurred via an inhibition of viral attachment to, but not internalization by, systemic macrophages through perturbation of signaling pathways involving RhoA/ROCK, AKT, and Rac. Pre-treatment using PI3Kδ-selective inhibitors prior to intravenous delivery of VV resulted in enhanced anti-tumor efficacy and significantly prolonged survival compared to delivery without PI3Kδ inhibition. These results indicate that effective intravenous delivery of oncolytic VV may be clinically achievable and could be useful in improving anti-tumor efficacy of oncolytic virotherapy., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Oncolytic viruses based on adenovirus type 5 (Ad5) have been developed as a new class of therapeutic agents for cancers that are resistant to conventional therapies. Clinical experience shows that these agents are safe, but virotherapy alone has not achieved long-term cure in cancer patients. The vast majority of oncolytic adenoviruses used in clinical trials to date have deletion of the E3B genes. It has been demonstrated that the antitumor potency of the E3B-deleted mutant (dl309) is inferior to adenovirus with E3B genes intact. Tumors treated with dl309 show markedly greater macrophage infiltration than E3B-intact adenovirus. However, the functional mechanisms for this were not previously known. Here, we demonstrate that deletion of E3B genes increases production of chemokines by monocytes after adenovirus infection and increases monocyte migration. The E3B 14,700-Da protein (E3B-14.7K) inhibits STAT1 function by preventing its phosphorylation and nuclear translocation. The STAT1 inhibitor, fludarabine, rescues the effect of E3B-14.7K deletion by downregulating target chemokine expression in human and murine monocytes and results in an enhanced antitumor efficacy with dl309 in vivo. These findings have important implications for clinical use of E3B-deleted oncolytic adenovirus and other E3B-deleted adenovirus vector-based therapy.

Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.

Purpose: The efficacy of oncolytic viruses depends on multiple actions including direct tumor lysis, modulation of tumor perfusion, and stimulation of tumor-directed immune responses. In this study, we investigated whether a sequential combination of immunologically distinct viruses might enhance antitumor efficacy through the induction of tumor-specific immunity and circumvention or mitigation of antiviral immune responses., Experimental Design: The Syrian hamster as an immune-competent model that supports replication of both adenovirus and vaccinia virus was evaluated in vitro and in vivo. The antitumor efficacy of either virus alone or sequential combination of the two viruses was examined in pancreatic and kidney cancer models. The functional mechanism of the regimen developed here was investigated by histopathology, immunohistochemistry staining, CTL assay, and T-cell depletion., Results: The Syrian hamster is a suitable model for assessment of oncolytic adenovirus and vaccinia virus. Three low doses of adenovirus followed by three low doses of vaccinia virus resulted in a superior antitumor efficacy to the reverse combination, or six doses of either virus alone, against pancreatic and kidney tumors in Syrian hamsters. A total of 62.5% of animals bearing either tumor type treated with the sequential combination became tumor-free, accompanied by the induction of effective tumor-specific immunity. This enhanced efficacy was ablated by CD3+ T-cell depletion but was not associated with humoral immunity against the viruses., Conclusion: These findings show that sequential treatment of tumors with oncolytic adenovirus and vaccinia virus is a promising approach for cancer therapy and that T-cell responses play a critical role., (2012 AACR.)

Oncolytic adenoviruses based on serotype 5 (Ad5) have several shortcomings, including the downregulation of its receptor in cancer cells, high prevalence of neutralizing antibodies and hepatotoxicity. Another adenoviral serotype, Ad11, could overcome these obstacles. Here, we show that human cancer cell lines express higher levels of the Ad11 receptor CD46, resulting in much better infectivity than Ad5. Surprisingly, only 36% (9/25) of the cell lines were more sensitive to Ad11- than to Ad5-mediated cytotoxicity. Investigations revealed that it was the transcription of Ad11 E1A, not CD46 expression or virus infectivity, which determined the cell's sensitivity to Ad11 killing. Ad11 E1A mRNA levels have an effect on viral DNA replication, structural protein synthesis and infectious particle production. To test the hypothesis that increased E1A transcription would lead to improved Ad11 replication in Ad5-sensitive (but Ad11-less sensitive) cells, two Ad11 mutants (Ad11-Ad5-P and Ad11-Ad5-EP) were constructed where either the E1A promoter or enhancer-promoter, respectively, was replaced by that of Ad5. Ad11-Ad5-EP demonstrated increased E1A mRNA levels and replication, together with enhanced oncolytic potency in vitro and in vivo. This effect was found in both the Ad5-sensitive and Ad11-sensitive cancer cells, broadening the range of tumors that could be effectively killed by Ad11-Ad5-EP.

Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.

The changes in cancer cell surface molecules and intracellular signaling pathways during tumorigenesis make delivery of adenovirus-based cancer therapies inefficient. Here we have identified carcinoembryonic antigen- related cell adhesion molecule 6 (CEACAM6) as a cellular protein that restricts the ability of adenoviral vectors to infect cancer cells. We have demonstrated that CEACAM6 can antagonize the Src signaling pathway, downregulate cancer cell cytoskeleton proteins, and block adenovirus trafficking to the nucleus of human pancreatic cancer cells. Similar to CEACAM6 overexpression, treatment with a Src-selective inhibitor significantly reduced adenovirus replication in these cancer cells and normal human epithelial cells. In a mouse xenograft tumor model, siRNA-mediated knockdown of CEACAM6 also significantly enhanced the antitumor effect of an oncolytic adenovirus. We propose that CEACAM6-associated signaling pathways could be potential targets for the development of biomarkers to predict the response of patients to adenovirus-based therapies, as well as for the development of more potent adenovirus-based therapeutics.

Pancreatic cancer is one of the most aggressive human tumors with a 5-year survival rate of only 3% and a striking resistance to chemotherapy and radiotherapy. The search for new therapeutic approaches includes strategies exploiting the deregulation of apoptotic pathways commonly found in cancer cells. The IAP proteins are inhibitors of apoptosis that have altered activity in numerous cancer types and are implicated in resistance to chemotherapy, and therefore are potentially interesting as therapeutic targets. We investigated alterations in the expression of IAPs and their inhibitors in pancreatic adenocarcinoma by using real-time PCR, in situ hybridization and immunohistochemistry. We found differential expression of various IAPs in this malignancy, and particularly we observed overexpression of cIAP-2, survivin, livin and XIAP. We also looked for correlations between the expression of IAPs and resistance to paclitaxel, doxorubicin, CDDP and 5-fluorouracil, and found that resistance to these drugs correlates most significantly with expression of cIAP-2. Using RNAi to downregulate these proteins we further confirmed that the levels of cIAP-2 and XIAP influence the response to the anti-cancer drugs, although only marginally for 5-FU. We conclude that anti-tumor strategies based on the inhibition of particular IAPs can be useful in targeting pancreatic adenocarcinoma.

Background & Aims: Markers to differentiate among pancreatic adenocarcinoma, chronic pancreatitis, and normal pancreas would be of significant clinical utility. This study was therefore designed to analyze the proteome of such specimens and identify new candidate proteins for differential diagnosis., Methods: A PowerBlot analysis with more than 900 well-characterized antibodies was performed with tissue specimens from patients with chronic pancreatitis, pancreatic adenocarcinoma, and normal pancreas. Differential expression of selected proteins was confirmed on a larger scale by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry using tissue arrays., Results: A total of 30 and 102 proteins showed significant deregulation between normal pancreas when compared with chronic pancreatitis and pancreatic adenocarcinoma, respectively, and although a substantial proportion were found similarly dysregulated in both chronic pancreatitis and pancreatic adenocarcinoma, several proteins were identified as potential disease-specific markers., Conclusions: A large number of proteins are differentially expressed in chronic pancreatitis and pancreatic adenocarcinoma compared with normal pancreas. Among these, expression analysis of UHRF1, ATP7A, and aldehyde oxidase 1 in combination could potentially provide a useful additional diagnostic tool for fine-needle aspirated or cytological specimens obtained during endoscopic investigations.

S100P is a member of the S100 family of calcium-binding proteins and there have been several recent reports of its overexpression in pancreatic ductal adenocarcinoma (PDAC). We have used Far Western screening and in vitro interaction assays to identify and confirm a novel target protein for S100P. We have named this protein S100PBPR, and shown that its interaction with S100P is dependent on Ca(2+) or Mg(2+). S100PBPR was found to localize to cell nuclei where S100P is also present, and the two proteins co-immunoprecipitate. By in situ hybridization, S100PBPR transcript was found in islet cells but not duct cells of the healthy pancreas. Both S100P and S100PBPR were detected by quantitative real-time polymerase chain reaction in pancreatic intraepithelial neoplasia (PanIN) and PDAC samples, and in situ hybridization revealed the presence of S100PBPR transcript in malignant PDAC cells. These data suggest that an interaction between S100P and S100PBPR may be involved in early pancreatic cancer. S100P was further investigated in PanIN lesions and immunohistochemical analysis showed its expression to correlate significantly with increasing grade of PanINs, being found as early as PanIN-1 with more prevalent expression in PanIN-2 and -3. These data suggest that S100P can be added to the genetic progression model for PDAC.

In order to expand our understanding of the molecular changes underlying the complex pathology of pancreatic malignancy, global gene expression profiling of pancreatic adenocarcinoma compared with normal pancreatic tissue was performed. Human cDNA arrays comprising 9932 elements were interrogated with fluorescence-labelled normal and adenocarcinoma samples (nine tumours, three normal pancreata, and three cell lines). The data were analysed for differential gene expression, which was confirmed by serial analysis of gene expression (SAGE), digital differential display (DDD) analysis, and immunohistochemistry for selected cases. The array data were filtered to produce lists of a total of 75 genes significantly up-regulated or down-regulated in pancreatic adenocarcinoma. Two of those showing the highest differential were members of the S100 family of Ca-binding proteins, namely S100P and S100A6, and therefore the S100 genes were studied in more detail. By immunohistochemical analysis of custom-built, pancreas-specific tissue arrays and commercially available, normal/cancer tissue arrays that included a wide variety of different tumour types, differential expression of S100P protein was found to be almost exclusive to pancreatic cancer. S100P could therefore represent a useful biomarker for pancreatic adenocarcinomas., (Copyright 2003 John Wiley & Sons, Ltd.)

The identification of deleterious mutations within candidate genes is a crucial step in the elucidation of the genetic bases of human disease. However, the significance of any base or amino acid change within a gene is unknown until detailed structural and functional analysis has been carried out. A potentially rapid way of identifying functionally important sites within a gene is to identify evolutionarily conserved regions. Mutations affecting such sites are assumed to be deleterious for the carrier. In this communication we generalize this approach and present a formal framework to assess whether a specific mutation is deleterious given sequence data from a set of homologues. We propose a score that takes into account the nature of the mutation, the conservation of the affected residue among the different species, and their phylogenetic relationships. Its performance is examined using published TP53 mutations and frequent polymorphic variants., (Copyright 2003 Wiley-Liss, Inc.)

A fertilization-induced increase in intracellular Ca2+ is responsible for initiating all of the events of egg activation. In mammals, the Ca2+ increase takes the form of a series of Ca2+ oscillations showing complex temporal and spatial properties. To understand the nature of these changes, we have investigated the expression patterns of the three isoforms of the inositol trisphosphate receptor (InsP3R) during oocyte maturation and preimplantation development. We find that mouse oocytes express mRNAs for all three InsP3R subtypes. Semiquantitative ratio reverse-transcriptase polymerase chain reaction shows that the type II isoform is the predominant message in mature oocytes, representing 67% of the InsP3R mRNA. In contrast, protein analysis reveals that the type I isoform accounts for all of the detectable InsP3R protein, despite representing only 20% of the InsP3R mRNA. The levels of InsP3R protein were examined to determine whether they correlated with the Ca2+ signaling events surrounding the fertilization process. Type I InsP3R protein increased during oocyte maturation and, in addition, within 8 h of fertilization underwent a dramatic decrease. During development to the blastocyst the level of type I InsP3R protein did not return to prefertilization levels and types II and III remained below our detection limit. The decrease in InsP3R protein after fertilization was found to correlate with a decrease in the sensitivity of InsP3-induced Ca2+ release. These studies show that the expression of InsP3R mRNA is developmentally regulated, that Ca2+ signaling at fertilization is mediated exclusively through the type I InsP3R, and that the InsP3R is downregulated after fertilization., (Copyright 1998 Academic Press.)

Flavodoxins synthesized by Azotobacter vinelandii strain UW 36 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter throococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem.J.277, 313-319]. Electrospray mass spectrometry gave M, values for the polypeptides of 19430 +/- 3 and 19533 +/- 5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (delta = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at delta = -142.1 p.p.m. and AvFld 2 at delta = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4(+)-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.