Pancreatic Cancer UK Grand Challenge: Developments and challenges for effective CAR T cell therapy for pancreatic ductal adenocarcinoma.

BACKGROUND: Death from pancreatic ductal adenocarcinoma (PDAC) is rising across the world and PDAC is predicted to be the second most common cause of cancer death in the USA by 2030. Development of effective biotherapies for PDAC are hampered by late presentation, a low number of differentially expressed molecular targets and a tumor-promoting microenvironment that forms both a physical, collagen-rich barrier and is also immunosuppressive. In 2017 Pancreatic Cancer UK awarded its first Grand Challenge Programme award to tackle this problem. The team plan to combine the use of novel CAR T cells with strategies to overcome the barriers presented by the tumor microenvironment. In advance of publication of those data this review seeks to highlight the key problems in effective CAR T cell therapy of PDAC and to describe pre-clinical and clinical progress in CAR T bio-therapeutics.

ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation.

The mechanisms of initiation of pancreatic ductal adenocarcinoma (PDAC) are still largely unknown. In the present study, we analysed the role of anterior gradient-2 (AGR2) in the earliest stages of pancreatic neoplasia. Immunohistochemical analysis of chronic pancreatitis (CP) and peritumoral areas in PDAC tissues showed that AGR2 was present in tubular complexes (TC) and early pancreatic intraepithelial neoplasia (PanINs). Moreover, AGR2 was also found in discrete subpopulations of non-transformed cells neighbouring these pre-neoplastic lesions. In primary cells derived from human patient-derived xenograft (PDX) model, flow-cytometry revealed that AGR2 was overexpressed in pancreatic cancer stem cells (CSC) compared with non-stem cancer cells. In LSL-KrasG12D;Pdx1-Cre (KC) mouse model Agr2 induction preceded the formation of pre-neoplastic lesions and their development was largely inhibited by Agr2 deletion in engineered LSL-KrasG12D;Pdx1-Cre; Agr2-/- mice. In vitro, AGR2 expression was stimulated by tunicamycin-induced endoplasmic reticulum (ER) stress in both KRAS wild-type normal pancreas cells, as well as in KRAS mutated pancreatic cancer cells and was essential for ER homoeostasis. The unfolded protein response proteins GRP78, ATF6 and XBP1s were found expressed in CP and PDAC peritumoral tissues, but in contrast to AGR2, their expression was switched off during TC and PanIN formation. Real-time PCR and ELISA analyses showed that ER stress induced a pro-inflammatory phenotype in pancreatic normal, cancer and stellate cells. Moreover, AGR2 expression was inducible by paracrine transfer of ER stress and pro-inflammation between different pancreatic cell types. Our findings demonstrate that AGR2 induced in ER-stressed and inflammatory pre-neoplastic pancreas is a potential marker of cancer progenitor cells with an important functional role in PDAC initiation.

Lister strain vaccinia virus with thymidine kinase gene deletion is a tractable platform for development of a new generation of oncolytic virus.

Vaccinia virus (VV) has many attractive characteristics as a potential cancer therapeutic. There are several strains of VV. The nonvaccine strain Western Reserve VV with deletion of both the thymidine kinase and the viral growth factor genes (known as WRDD) has been reported as the most potent tumor-targeted oncolytic VV. Other strains, such as the European vaccine Lister strain, are largely untested. This study evaluated the antitumor potency and biodistribution of different VV strains using in vitro and in vivo models of cancer. Lister strain virus with thymidine kinase gene deletion (VVΔTK) demonstrated superior antitumor potency and cancer-selective replication in vitro and in vivo, compared with WRDD, especially in human cancer cell lines and immune-competent hosts. Further investigation of functional mechanisms revealed that Lister VVΔTK presented favorable viral biodistribution within the tumors, with lower levels of proinflammatory cytokines compared with WRDD, suggesting that Lister strain may induce a diminished host inflammatory response. This study indicates that the Lister strain VVΔTK may be a particularly promising VV strain for the development of the next generation of tumor-targeted oncolytic therapeutics.

The oncolytic adenovirus AdΔΔ enhances selective cancer cell killing in combination with DNA-damaging drugs in pancreatic cancer models.

Pancreatic adenocarcinomas are aggressive and frequently develop resistance to all current therapies. Replication-selective adenoviruses can overcome resistance to chemotherapeutics through their sensitizing effects on drug-induced cell killing. We previously found that adenovirus deleted in the anti-apoptotic E1B19K gene enhanced gemcitabine-induced apoptotis. Here we demonstrate that our engineered double-deleted AdΔΔ mutant (deleted in the pRb-binding E1ACR2 region and E1B19K) selectively replicates and enhances cell killing in combination with DNA-damaging cytotoxic drugs in pancreatic cancer cells. Combinations of AdΔΔ with gemcitabine, irinotecan or cisplatin resulted in two- to fourfold decreases in EC(50) (half maximal effective concentration) values and was more efficent than similar combinations with wild-type virus, the dl1520 (ONYX-015) and dl922-947 mutants. AdΔΔ replication was impaired in normal bronchial human epithelial cells and did not sensitize the cells to drugs. Gemcitabine-insensitive AsPC-1, BxPC-3 and PANC-1 cells were efficiently killed by irinotecan in combination with AdΔΔ. Suboptimal doses of AdΔΔ and gemcitabine significantly prolonged time to tumor progression in two human pancreatic tumor xenograft in vivo models, PT45 and SUIT-2. We conclude that AdΔΔ has low toxicity to normal cells while potently sensitizing pancreatic cancer cells to DNA-damaging drugs, and holds promise as an improved therapeutic strategy for pancreatic cancer.

An oncolytic adenovirus defective in pRb-binding (dl922-947) can efficiently eliminate pancreatic cancer cells and tumors in vivo in combination with 5-FU or gemcitabine.

Pancreatic adenocarcinoma has a poor prognosis and frequently develops resistance to standard chemotherapeutics. Oncolytic adenoviruses represent a promising approach to overcome treatment resistance. The replication-selective dl922-947 adenovirus, defective in pRb binding, targets cancers with deregulated cell cycle control, such as the majority of pancreatic tumors. Cell killing efficacy was higher for dl922-947 than for adenovirus type 5 (Ad5) and the clinically approved dl1520 in pancreatic cancer cells with K-ras, p16 and p53 mutations. Combinations of dl922-947 and 5-fluorouracil or gemcitabine (2'2'-difluoro-2-deoxytidine) resulted in strong synergistic cell killing in Suit-2 and the highly drug- and virus-resistant Hs766T cells. Viral uptake increased in response to drugs, but was independent of the expression levels of the viral attachment receptor coxsackie and adenovirus receptor (CAR), whereas expression levels of the internalization receptors α(v)ß(3)- and α(v)ß(5)-integrins were increased. Early viral E1A expression was potently induced with drugs contributing to the synergistic effects. The dl922-947 mutant was more efficacious than Ad5 in vivo in Hs766T and Suit-2 xenograft models. In combination with gemcitabine, median survival was further prolonged. We demonstrate that dl922-947 is highly efficacious in pancreatic cancers and conclude that oncolytic adenoviruses harboring the E1ACR2 deletion have great potential for development into future clinical candidates for pancreatic cancer.

Lister strain vaccinia virus, a potential therapeutic vector targeting hypoxic tumours.

Hypoxia contributes to the aggressive and treatment-resistant phenotype of pancreatic ductal adenocarcinoma. Oncolytic vaccinia virus has potential as an anti-tumour agent, but the ability to lyse hypoxic tumour cells is vital for clinical efficacy. We hypothesized that unique aspects of the poxvirus life cycle would protect it from attenuation in hypoxic conditions. We characterized and compared the viral protein production, viral replication, cytotoxicity and transgene expression of Lister strain vaccinia virus in a panel of pancreatic cancer cell lines after exposure to normoxic or hypoxic conditions. Viral protein production was not affected by hypoxia, and high viral titres were produced in both normoxic and hypoxic conditions. Interestingly, there was a 3.5-fold (P<0.001) and 20-fold (P<0.0001) increase in viral cytotoxicity for CFPac1 and MiaPaca2 cell lines, respectively, in hypoxic conditions. Cytotoxicity was equivalent in the remaining cell lines. Levels of transgene expression (luciferase reporter gene) from the vaccinia viral vector were comparable, regardless of the ambient oxygen concentration. The present study suggests that the vaccinia virus is a promising vector for targeting pancreatic cancer and potentially other hypoxic tumour types.

Lister strain of vaccinia virus armed with endostatin-angiostatin fusion gene as a novel therapeutic agent for human pancreatic cancer.

Survival after pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (i.v.) and intratumoral (i.t.) administrations. The expression of the endostatin-angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus showed significant antitumor potency in vivo against the Suit-2 model by i.t. administration. This study suggests that the novel Lister strain of vaccinia virus armed with the endostatin-angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer.

Update on the molecular pathogenesis of pancreatic tumors other than common ductal adenocarcinoma.

PURPOSE: Although ductal adenocarcinoma is the most common and well known pancreatic tumor type, other distinct epithelial neoplasms affecting the pancreas that show different symptoms, biological behaviors and outcomes are becoming more frequently recognized and documented. Pancreatic epithelial tumors may be separated into ductal and nonductal neoplasms. The former group includes pancreatic ductal adenocarcinoma, intraductal papillary-mucinous tumor, mucinous cystic tumor and serous cystic tumor. The latter group includes pancreatic endocrine tumor, pancreatic acinar cell carcinoma, pancreatoblastoma and solid-pseudopapillary tumor. The aim of this review is to summarize recently acquired knowledge regarding the molecular characterization of these uncommon pancreatic epithelial neoplasms. RECENT FINDINGS: Molecular studies of uncommon pancreatic epithelial tumors suggest that the different morphological entities are associated with distinct molecular profiles, highlighting the involvement of different molecular pathways leading to the development of each subtype of pancreatic neoplasm. CONCLUSION: The correct classification of rare pancreatic epithelial tumors and the identification of their characteristic molecular aspects is the fundamental starting point in identifying novel diagnostic molecular tools and new targets for innovative therapeutic strategies.

Targeted therapies for pancreatic cancer.

INTRODUCTION: Pancreatic cancer is a devastating malignancy and a leading cause of cancer mortality. Furthermore, early diagnosis represents a serious hurdle for clinicians, as symptoms are non-specific and usually manifest in advanced, treatment-resistant stages of the disease. SOURCES OF DATA: Here, we review the rationale and progress of targeted therapies currently under investigation. AREAS OF AGREEMENT: At present, chemoradiation regimes are administered palliatively, and produce only marginal survival benefits, underscoring a desperate need for more effective treatment modalities. AREAS OF CONTROVERSY: Questions have been raised as to whether erlotinib, the only targeted therapy to attain a statistically significant increase in median survival, is cost-effective. GROWING POINTS: The last decade of research has provided us with a wealth of information regarding the molecular nature of pancreatic cancer, leading to the identification of signalling pathways and their respective components which are critical for the maintenance of the malignant phenotype. AREAS TIMELY FOR DEVELOPING RESEARCH: These proteins thus represent ideal targets for novel molecular therapies which embody an urgently needed novel treatment strategy.

Yes-associated protein (YAP) functions as a tumor suppressor in breast.

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

Targeting sodium/iodide symporter gene expression for estrogen-regulated imaging and therapy in breast cancer.

Expression of the sodium iodide symporter (hNIS) has been detected in breast cancer tissue, but frequently, not at the levels necessary to mediate (131)I accumulation. Transducing the hNIS gene into breast cancer cells with adenovirus could be a tractable strategy to render breast cancer susceptible to radioiodide therapy. We constructed the replication-incompetent virus, AdSERE, in which an estrogen-responsive promoter directs the expression of hNIS. In vitro, we demonstrate that AdSERE mediates hNIS expression and iodide uptake in ER+ breast cancer cells. In vivo, we show that AdSERE-infected ER+ tumors can be imaged due to tracer accumulation; in addition, AdSERE in combination with therapeutic doses of (131)I suppresses tumor growth.

Oncolytic adenoviral mutants induce a novel mode of programmed cell death in ovarian cancer.

Oncolytic adenoviral mutants have considerable activity in ovarian cancer. However, the mechanisms by which they induce cell death remain uncertain. dl922-947, which contains a 24 bp deletion in E1A CR2, is more potent than both E1A wild-type adenoviruses and the E1B-55K deletion mutant dl1520 (Onyx-015). We investigated the mode of death induced by three E1A CR2-deleted replicating adenoviruses in models of ovarian cancer and also the importance of E3 11.6 (adenovirus death protein) in determining this mode of death. Ovarian cancer cells were infected with dl922-947 (E3 11.6+) and dlCR2 (E3 11.6-). We also generated dlCR2 tSmac, which also encodes the gene for processed Smac/DIABLO. Classical apoptosis does not occur in adenoviral cell death and there is no role for mitochondria. Expression of Smac/DIABLO does not enhance cytotoxicity nor increase apoptotic features. A role for cathepsins and lysosomal membrane permeability was excluded. Autophagy is induced, but is not the mode of death and may act as a cell survival mechanism. There is no evidence of pure necrosis, while the presence of E3 11.6 does not modulate the mode or extent of cell death. Thus, E1A CR2-deleted oncolytic adenoviral cytotoxicity in ovarian cancer may define a novel mode of programmed cell death.

Genome-wide DNA copy number analysis in pancreatic cancer using high-density single nucleotide polymorphism arrays.

To identify genomic abnormalities characteristic of pancreatic ductal adenocarcinoma (PDAC) in vivo, a panel of 27 microdissected PDAC specimens were analysed using high-density microarrays representing approximately 116 000 single nucleotide polymorphism (SNP) loci. We detected frequent gains of 1q, 2, 3, 5, 7p, 8q, 11, 14q and 17q (> or =78% of cases), and losses of 1p, 3p, 6, 9p, 13q, 14q, 17p and 18q (> or =44%). Although the results were comparable with those from array CGH, regions of those genetic changes were defined more accurately by SNP arrays. Integrating the Ensembl public data, we have generated 'gene' copy number indices that facilitate the search for novel candidates involved in pancreatic carcinogenesis. Copy numbers in a subset of the genes were validated using quantitative real-time PCR. The SKAP2/SCAP2 gene (7p15.2), which belongs to the src family kinases, was most frequently (63%) amplified in our sample set and its recurrent overexpression (67%) was confirmed by reverse transcription-PCR. Furthermore, fluorescence in situ hybridization and in situ RNA hybridization analyses for this gene have demonstrated a significant correlation between DNA copy number and mRNA expression level in an independent sample set (P<0.001). These findings indicate that the dysregulation of SKAP2/SCAP2, which is mostly caused by its increased gene copy number, is likely to be associated with the development of PDAC.

E1A-expressing adenoviral E3B mutants act synergistically with chemotherapeutics in immunocompetent tumor models.

The majority of clinical trials evaluating replication-selective oncolytic adenoviruses utilized mutants with immunomodulatory E3B genes deleted, likely contributing to the attenuated efficacy. We investigated whether an intact immune response could contribute to the observed improved efficacy in response to combinations with chemotherapeutics. Seven carcinoma cell lines were evaluated by combining viral mutants; dl309 (DeltaE3B), dl704 (DeltaE3gp19K), dl312 (DeltaE1A) or wild-type Ad5 with the commonly used clinical drugs cisplatin and paclitaxel. Synergistic effects on cell death were determined by generation of combination indexes in cultured cells. In vivo tumor growth inhibition was achieved by virotherapy alone and was most efficacious with wild-type virus and least with the DeltaE3B mutant. Significantly higher efficacy was observed when the viruses were combined with drugs. The greatest enhancement of tumor inhibition was in combination with the DeltaE3B mutant restoring potency to that of Ad5 wild-type levels, observed only in animals with intact immune response. Increases in infectivity, viral gene expression and replication were identified as potential mechanisms contributing to the synergistic effects. Our results suggest that the attenuation of DeltaE3B mutants can be overcome by low doses of chemotherapeutics only in the presence of an intact immune response indicating a role for T-cell-mediated functions.

Progress and prospects: gene therapy clinical trials (part 2).

This is the second part of a review summarizing progress and prospects in gene therapy clinical research. Twenty key diseases/strategies are succinctly described and commented on by leaders in the field. This part includes clinical trials for skin diseases, neurological disorders, HIV/AIDS, ornithine transcarbamylase deficiency, alpha(1)-antitrypsin deficiency, haemophilia and cancer.

Identification of genetic alterations in pancreatic cancer by the combined use of tissue microdissection and array-based comparative genomic hybridisation.

Pancreatic ductal adenocarcinoma (PDAC) is characterised pathologically by a marked desmoplastic stromal reaction that significantly reduces the sensitivity and specificity of cytogenetic analysis. To identify genetic alterations that reflect the characteristics of the tumour in vivo, we screened a total of 23 microdissected PDAC tissue samples using array-based comparative genomic hybridisation (array CGH) with 1 Mb resolution. Highly stringent statistical analysis enabled us to define the regions of nonrandom genomic changes. We detected a total of 41 contiguous regions (>3.0 Mb) of copy number changes, such as a genetic gain at 7p22.2-p15.1 (26.0 Mb) and losses at 17p13.3-p11.2 (13.6 Mb), 18q21.2-q22.1 (12.0 Mb), 18q22.3-q23 (7.1 Mb) and 18q12.3-q21.2 (6.9 Mb). To validate our array CGH results, fluorescence in situ hybridisation was performed using four probes from those regions, showing that these genetic alterations were observed in 37-68% of a separate sample set of 19 PDAC cases. In particular, deletion of the SEC11L3 gene (18q21.32) was detected at a very high frequency (13 out of 19 cases; 68%) and in situ RNA hybridisation for this gene demonstrated a significant correlation between deletion and expression levels. It was further confirmed by reverse transcription-PCR that SEC11L3 mRNA was downregulated in 16 out of 16 PDAC tissues (100%). In conclusion, the combination of tissue microdissection and array CGH provided a valid data set that represents in vivo genetic changes in PDAC. Our results raise the possibility that the SEC11L3 gene may play a role as a tumour suppressor in this disease.

Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility.

BACKGROUND: Previously, proteomic methods were applied to characterise differentially expressed proteins in microdissected pancreatic ductal adenocarcinoma cells. AIMS: To report that CapG and a related protein, gelsolin, which have established roles in cell motility, are overexpressed in metastatic pancreatic cancer; and to describe their pattern of expression in pancreatic cancer tissue and their effect on cell motility in pancreatic cancer cell lines. METHODS: CapG was identified by mass spectrometry and immunoblotting. CapG and gelsolin expression was assessed by immunohistochemical analysis on a pancreatic cancer tissue microarray and correlated with clinical and pathological parameters. CapG and gelsolin levels were reduced using RNA interface in Suit-2, Panc-1 and MiaPaCa-2 cells. Cell motility was assessed using modified Boyden chamber or wound-healing assays. RESULTS: Multiple isoforms of CapG were detected in pancreatic cancer tissue and cell lines. Immunohistochemical analysis of benign (n = 44 patients) and malignant (n = 69) pancreatic ductal cells showed significantly higher CapG staining intensity in nuclear (p<0.001) and cytoplasmic (p<0.001) compartments of malignant cells. Similarly, gelsolin immunostaining of benign (n = 24 patients) and malignant (n = 68 patients) pancreatic ductal cells showed higher expression in both compartments (both p<0.001). High nuclear CapG was associated with increased tumour size (p = 0.001). High nuclear gelsolin was associated with reduced survival (p = 0.01). Reduction of CapG or gelsolin expression in cell lines by RNAi was accompanied by significantly impaired motility. CONCLUSIONS: Up regulation of these actin-capping proteins in pancreatic cancer and their ability to modulate cell motility in vitro suggest their potentially important role in pancreatic cancer cell motility and consequently dissemination.

Sperm-associated antigen 1 is expressed early in pancreatic tumorigenesis and promotes motility of cancer cells.

Sperm-associated antigen 1 (SPAG1) was recently identified in a rare form of infertility where anti-SPAG1 antibodies derived from the serum of an infertile woman were reported to cause sperm agglutination. Except for its expression and potential role in spermatogenesis, the function of SPAG1 is completely unknown. The unexpected finding of high levels of SPAG1 expression in pancreatic adenocarcinoma compared to normal pancreatic tissue in our previous cDNA array experiments prompted us to look in more detail at the expression and role of this gene in a panel of normal and malignant human tissues as well as in a larger series of pancreatic cancer specimens. We have generated an SPAG1-specific monoclonal antibody and showed high levels of SPAG1 protein in testis and in a large proportion of pancreatic ductal adenocarcinomas (PDAC). In the latter, SPAG1 expression was predominantly cytoplasmic and confined to malignant cells. Furthermore, the extent and intensity of SPAG1 expression was shown to be associated with stage and tumour nodal status, while analysis of precursor lesions, pancreatic intraepithelial neoplasias (PanINs), demonstrated its increased immunoreactivity with increasing PanIN grade, suggesting that SPAG1 is a novel marker of PDAC progression. Immunocytochemical analysis demonstrated colocalization of SPAG1 with microtubules, and their association was confirmed by co-immunoprecipitation; subsequent motility assays further substantiated a potential role of SPAG1 in cancer cell motility. Combined with the finding of its early expression in PDAC development, our data suggest that SPAG1 could contribute to the early spread and poor prognosis of pancreatic adenocarcinoma.

Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the beta4 integrin and the PI3k pathway.

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 alpha(6)beta(4) 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.