Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease in humans. Transforming growth factor-β (TGF-β) signaling plays an important role in PDAC progression, as indicated by the fact that Smad4, which encodes a central signal mediator downstream from TGF-β, is deleted or mutated in 55% and the type II TGF-β receptor (Tgfbr2) gene is altered in a smaller subset of human PDAC. Pancreas-specific Tgfbr2 knockout mice have been generated, alone or in the context of active Kras (Kras^G12D) expression, using the Cre-loxP system driven by the endogenous Ptf1a (pancreatic transcription factor-1a) locus. Pancreas-selective Tgfbr2 knockout alone gave no discernable phenotype in 1.5 yr. Pancreas-specific Kras^G12D activation alone essentially generated only intraepithelial neoplasia within 1 yr. In contrast, the Tgfbr2 knockout combined with Kras^G12D expression developed well-differentiated PDAC with 100% penetrance and a median survival of 59 d. Heterozygous deletion of Tgfbr2 with Kras^G12D expression also developed PDAC, which indicated a haploinsufficiency of TGF-β signaling in this genetic context. The clinical and histopathological manifestations of the combined Kras^G12D expression and Tgfbr2 knockout mice recapitulated human PDAC. The data show that blockade of TGF-β signaling and activated Ras signaling cooperate to promote PDAC progression.

Dysregulated DNA methylation followed by abnormal gene expression is an epigenetic hallmark in cancer. DNA methylation is catalyzed by DNA methyltransferases, and the aberrant expression or mutations of DNA methyltransferase genes are found in human neoplasm. The enzymes for demethylating 5‐methylcytosine were recently identified, and the biological significance of DNA demethylation is a current focus of scientific attention in various research fields. Ten–eleven translocation (TET) proteins have an enzymatic activity for the conversion from 5‐methylcytosine to 5‐hydroxymethylcytosine (5‐hmC), which is an intermediate of DNA demethylation. The loss‐of‐function mutations of TET2 gene were reported in myeloid malignancies, suggesting that impaired TET‐mediated DNA demethylation could play a crucial role in tumorigenesis. It is still unknown, however, whether DNA demethylation is involved in biological properties in solid cancers. Here, we show the loss of 5‐hmC in a broad spectrum of solid tumors: for example, a significant reduction of 5‐hmC was found in 72.7% of colorectal cancers (CRCs) and 75% of gastric cancers compared to background tissues. TET1 expression was decreased in half of CRCs, and a large part of them was followed by the loss of 5‐hmC. These findings suggest that the amount of 5‐hmC in tumors is often reduced via various mechanisms, including the downregulation of TET1. Consistently, in the in vitro experiments, the downregulation of TET1 was clearly induced by oncogene‐dependent cellular transformation, and loss of 5‐hmC was seen in the transformed cells. These results suggest the critical roles of aberrant DNA demethylation for oncogenic processes in solid tissues. (Cancer Sci 2012; 103: 670–676)

Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal neoplasms, is characterized by an expanded stroma with marked fibrosis (desmoplasia). We previously generated pancreas epithelium–specific TGF-β receptor type II (Tgfbr2) knockout mice in the context of Kras activation (mice referred to herein as Kras+Tgfbr2^KO mice) and found that they developed aggressive PDAC that recapitulated the histological manifestations of the human disease. The mouse PDAC tissue showed strong expression of connective tissue growth factor (Ctgf), a profibrotic and tumor-promoting factor, especially in the tumor-stromal border area, suggesting an active tumor-stromal interaction. Here we show that the PDAC cells in Kras+Tgfbr2^KO mice secreted much higher levels of several Cxc chemokines compared with mouse pancreatic intraepithelial neoplasia cells, which are preinvasive. The Cxc chemokines induced Ctgf expression in the pancreatic stromal fibroblasts, not in the PDAC cells themselves. Subcutaneous grafting studies revealed that the fibroblasts enhanced growth of PDAC cell allografts, which was attenuated by Cxcr2 inhibition. Moreover, treating the Kras+Tgfbr2^KO mice with the CXCR2 inhibitor reduced tumor progression. The decreased tumor progression correlated with reduced Ctgf expression and angiogenesis and increased overall survival. Taken together, our data indicate that tumor-stromal interactions via a Cxcr2-dependent chemokine and Ctgf axis can regulate PDAC progression. Further, our results suggest that inhibiting tumor-stromal interactions might be a promising therapeutic strategy for PDAC.

Background: Methods: Results: Conclusions:

PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent prostate cancers. PSES shows strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4PSESE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer PSES and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of PSES being very weak in nonprostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in PSA/PSMA-positive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv s.c. tumors were induced in nude mice and treated with AdE4PSESE1a via intratumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intratumoral injection. These data show that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer.