Frequent genomic copy number gain and overexpression of GATA-6 in pancreatic carcinoma

Multiple genetic alterations are well recognized as contributing to pancreatic carcinogenesis, although the finding of recurrent copy number changes indicates additional targets remain to be found. The objective of this study was to identify novel targets of genetic alteration that contribute to pancreatic cancer development or progression. We used Representational Oligonucleotide Microarray Analysis (ROMA) to identify copy number changes in pancreatic cancer xenografts, and validated these findings using FISH, quantitative PCR, Western blotting and immunohistochemical labeling. With this approach, we identified a 0.36-Mb amplification at 18q11.2 containing two known genes, GATA-6 and cTAGE1. Using a cutoff value of 3.0 fold compared to haploid controls, copy number gain or amplification was confirmed in 4 of 42 (9.5%) pancreatic carcinomas analyzed. Combined genetic and transcriptional analyses showed consistent overexpression of GATA-6 in all carcinomas with 18q11.2 gain, as well as in the majority of pancreatic cancers examined (17 of 30 cancers, 56.7%) that did not have gain of this region. By contrast, overexpression of cTAGE1 was rare in these same cancers suggesting GATA-6 is the true target of this copy number increase. GATA-6 mRNA overexpression corresponded to robust nuclear protein expression in cancer cell lines and resected tissues consistent with its role as a transcription factor. Intense nuclear labeling was significantly increased in PanIN-3 lesions and infiltrating carcinomas compared to normal duct epithelium (p < 0.000001 and p < 0.003, respectively). Forced overexpression of GATA6 in MiaPaca2 cells resulted in increased proliferation and growth in soft-agar. Gain and overexpression of the development-related transcription factor GATA-6 may play an important and hitherto unrecognized role in pancreatic carcinogenesis

Copy number alterations in pancreatic cancer identify recurrent PAK4 amplification

Pancreatic cancer is one of the most lethal of all cancers. The median survival is 6 months, and less than 5% of those diagnosed survive 5-years. Recurrent genetic deletions and amplifications in 73 pancreatic adenocarcinomas, the largest sample set analyzed to date for pancreatic cancer, were defined using comparative genomic hybridization The recurrent genetic alterations identified target a number of previously well-characterized genes, as well as regions that contain possible new oncogenes and tumor suppressor genes. We have focused on chromosome 19q13, a region frequently found amplified in pancreatic cancer, and demonstrate how boundaries of common regions of mutation can be mapped, and how a gene, in this case PAK4 amplified on chromosome19q13, can be functionally validated. We show that although the PAK4 gene is not activated by mutation in cell lines with gene amplification, an oncogenic form of the KRAS2 gene is present in these cells, and oncogenic KRAS2 can activate PAK4. In fact in the three samples we identified with PAK4 gene amplification, the KRAS2 gene was activated and genomically amplified. The kinase activity of the PAK4 protein is significantly higher in cells with genomic amplification as compared to cells without amplification. Our study demonstrates the utility of analyzing copy number data in a large set of neoplasms to identify genes involved in cancer. We have generated a useful dataset which will be particularly useful for the pancreatic cancer community as efforts are undertaken to sequence the pancreatic cancer genome

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

Unresolved endoplasmic reticulum stress engenders immune-resistant, latent pancreatic cancer metastases

The majority of patients with pancreatic ductal adenocarcinoma (PDA) develop metastatic disease after resection of their primary tumor. We found that livers from patients and mice with PDA harbor single, disseminated cancer cells (DCCs) lacking expression of cytokeratin-19 (CK19) and major histocompatibility complex class I (MHCI). We created a mouse model to determine how these DCCs develop. Intra-portal injection of immunogenic PDA cells into pre-immunized mice seeded livers only with single, non-replicating DCCs that were CK19(-) and MHCI(-) The DCCs exhibited an endoplasmic reticulum (ER) stress response but, paradoxically lacked both inositol-requiring enzyme 1alpha activation and expression of the spliced form of transcription factor XBP1 (XBP1s). Inducible expression of XBP1s in DCCs, in combination with T cell-depletion, stimulated the outgrowth of macro-metastatic lesions that expressed CK19 and MHCI. Thus, unresolved ER stress enables DCCs to escape immunity and establish latent metastases

A Quantitative System for Studying Metastasis Using Transparent Zebrafish

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF[superscript V600E];p53[superscript −/−] fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, μ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.National Institutes of Health (U.S.) (Directors New Innovator Award DP2CA186572 and K08AR055368)Melanoma Research Alliance (Young Investigator Award)American Association for Cancer Research/American Society of Clinical Oncology (Young Investigator Award)Memorial Sloan-Kettering Cancer Center. Alan and Sandra Gerry Metastasis Research InitiativeHoward Hughes Medical Institut

A Six-Gene Signature Predicts Survival of Patients with Localized Pancreatic Ductal Adenocarcinoma

Jen Jen Yeh and colleagues developed and validated a six-gene signature in patients with pancreatic ductal adenocarcinoma that may be used to better stage the disease in these patients and assist in treatment decisions

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype

Neoantigen quality predicts immunoediting in survivors of pancreatic cancer.

Cancer immunoediting1 is a hallmark of cancer2 that predicts that lymphocytes kill more immunogenic cancer cells to cause less immunogenic clones to dominate a population. Although proven in mice1,3, whether immunoediting occurs naturally in human cancers remains unclear. Here, to address this, we investigate how 70 human pancreatic cancers evolved over 10 years. We find that, despite having more time to accumulate mutations, rare long-term survivors of pancreatic cancer who have stronger T cell activity in primary tumours develop genetically less heterogeneous recurrent tumours with fewer immunogenic mutations (neoantigens). To quantify whether immunoediting underlies these observations, we infer that a neoantigen is immunogenic (high-quality) by two features-'non-selfness'  based on neoantigen similarity to known antigens4,5, and 'selfness'  based on the antigenic distance required for a neoantigen to differentially bind to the MHC or activate a T cell compared with its wild-type peptide. Using these features, we estimate cancer clone fitness as the aggregate cost of T cells recognizing high-quality neoantigens offset by gains from oncogenic mutations. With this model, we predict the clonal evolution of tumours to reveal that long-term survivors of pancreatic cancer develop recurrent tumours with fewer high-quality neoantigens. Thus, we submit evidence that that the human immune system naturally edits neoantigens. Furthermore, we present a model to predict how immune pressure induces cancer cell populations to evolve over time. More broadly, our results argue that the immune system fundamentally surveils host genetic changes to suppress cancer