Referee Comments: Referee 1

Reviewer 1's Review

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Pancreatic ductal adenocarcinoma (PDA) is a deadly malignancy that is marked by resistance to chemotherapeutic intervention. Understanding the signaling pathways that are activated in this tumor type may provide new opportunities for therapeutic intervention. This manuscript by Pasca di Magliano and colleagues explores whether the canonical Wnt signaling pathway is commonly activated in PDA, whether the pathway is stimulated by hedgehog signaling, and the importance of the Wnt pathway in the proliferation and survival of PDA cells. Thus, this manuscript addresses a topic of great importance and should be of interest to a broad readership.

The authors present data from several complementary approaches that support their hypothesis that the canonical Wnt/β-catenin signaling axis is important in this malignancy. They utilize human tumor samples, PDA cell lines, mouse models, and gene expression array data to generate a compendium of data that coalesce around their hypothesis. While these data all support the central argument of the authors, and are generally nicely presented, there are a few issues that should be addressed before this manuscript is suitable for publication and discussion by the larger scientific community.

1) The authors show that 13% of PDA samples show nuclear β-catenin staining indicative of pathway activation. However, the majority of samples (65%) show either absence of membrane staining and/or cytoplasmic staining. No connection is made to determine E-cadherin status in these samples, and the absence of E-cadherin may release β-catenin from the membrane but not result in activation of the Wnt/β-catenin transcriptional program. This should be explored, at least in a subset of the samples, in order to be able to conclude that the pathway is activated.

2) The gene expression array experiments show increased expression of several pathway components and target genes. However, the target genes highlighted respond to several signals, and their elevated mRNA levels do not necessarily reflect activation of the pathway. Most interesting perhaps are the DICKKOPF genes as these have been suggested to create a negative feedback loop. Verification of the DICKKOPF genes by QRT-PCR would greatly support the conclusions drawn from these data.

The experimental methods used for the array experiments are not described in the manuscript. This is important as PDAs have a significant stromal component that would need to be separated from the cancer cells, by laser capture dissection or some other method, before the performance of RNA isolation. Description of these methods is needed.

3) The data from the KRas mouse model are convincing and the use of the TOPGal transgenic line is clever. However, a better control for these experiments would have been a Pdx-cre; TOPGal cross as opposed to TOPGal alone. While this experiment cannot be performed in the time frame for revision, I would encourage the authors to consider this and add it to the record at a later date.

4) Impressive data are presented in PDA cell lines demonstrating activation of the Wnt/β-catenin signaling axis in these cell lines, and the involvement of this pathway in the proliferation and survival of these cells. However, interpretation of these experiments (particularly the data in figures 2A and 2B) is hampered by the absence of a suitable negative control such as human pancreatic duct epithelial cells (PDCs). Thus it is impossible to know how the levels of pathway activation compare to untransformed pancreatic cells, or whether these findings are simply cell culture induced artifacts. Repetition of the experiments shown in figure 2 using murine PDA cell lines, and using murine PDCs as a control, may be an alternative approach. Also, demonstration of increased binding to the promoters of a subset of target genes using chromatin immunoprecipitation would have been preferred (or at least a nice complement) to the luciferase assays.

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N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.