PodcastGenomic Screening

Science Signaling Podcast: 11 November 2008

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Science Signaling  11 Nov 2008:
Vol. 1, Issue 45, pp. pc11
DOI: 10.1126/scisignal.145pc11


This is a conversation with two of the authors of a Research Article published in the 11 November issue of Science Signaling. They explain how combining a functional genetics screen with proteomics data provides new insight into Wnt signaling in a particular human cell line.

(Length: 10 min; file size: 4.84 MB; file format: mp3; location: http://podcasts.aaas.org/science_signaling/ScienceSignaling_081111.mp3)

Technical Details

Length: 10 min

File size: 4.84 MB

File Format: mp3

RSS Feed: http://stke.sciencemag.org/rss/podcast.xml

Download Podcast: http://podcasts.aaas.org/science_signaling/ScienceSignaling_081111.mp3

Educational Details

Learning Resource Type: Audio

Context: High school upper division 11-12, undergraduate lower division 13-14, undergraduate upper division 15-16, graduate, professional, general public and informal education

Intended Users: Teacher, learner

Intended Educational Use: Learn, teach

Discipline: Cell Biology, Genomics, Proteomics, Molecular Biology

Keywords: Science Signaling, signal transduction, genetic screen, Wnt, Wingless, RNAi, proteomics, colon cancer


Host – Annalisa VanHookWelcome to the Science Signaling Podcast for November 11th, 2008. I’m Annalisa VanHook. In this episode we’ll be discussing a screen designed to identify regulators of Wnt signaling (1). This screen was innovative because it combined a rigorous functional screen with proteomics data to identify proteins that contribute to Wnt signaling in a specific cell type—colon cancer cells.

This screen was carried out by Ben Major, a postdoctoral fellow in Randy Moon’s lab at the University of Washington. First, I spoke with Dr. Moon to get an overview of Wnt signaling. And, I asked him, “Why study the Wnt signaling pathway in the first place?”

Interviewee – Randall MoonIt actually goes back to work of over 20 years ago, when it was discovered, in two different systems, that Wnts were going to be extremely exciting. And so, in one context a Wnt was discovered as a gene that’s involved in Drosophila development, and when it was knocked out they ended up with a funny, funny phenotype so it was called the wingless gene. So, at that point we knew it was involved in development. And then, at about the same time the same gene was discovered in mouse, but it was discovered as a gene that causes mammary tumors. And so, at that point we knew that this same gene was somehow involved in both development and cancer, which raised considerable interest in it.

So, this pathway has been under investigation for at least 20 years because of its involvement in both development and cancer and as well as other diseases. And, people have been studying it by a variety of techniques—genetics and biochemistry—and basically bootstrapping their way into any new technique or technology that would yield some insight to it. And, the amazing thing about this pathway, despite its being studied for over 20 years, is that people are continually finding new components, so we haven’t exhausted it.

So, that brought us to the point where we ultimately just sat down and said, “Okay, what do we know about the Wnt signaling pathway, in any one given cell type.” And, the surprising answer, I think, was that by and large we didn’t really know what the pathway was in any one cell type; we knew sort of a composite picture of what it looked like in general. When you took one component from this cell and one component from that cell and realized people have been studying it in so many different cell types and organisms that all you could do was make a big composite picture. So, we decided that it would be essential to define this pathway in one cell type and do it as completely as humanly possible—both at the level of identifying genes that were functionally linked to the pathway and also identifying proteins that participate in the signal transduction itself.

Interviewer – Annalisa VanHookThat was Dr. Moon giving us a brief overview of research on the Wnt signaling pathway. Next, I spoke with his colleague, Ben Major, about the screen he performed. Hi, Dr. Major.

Interviewee – Ben MajorHi.

Interviewer – Annalisa VanHookSince the Wnt pathway is so well-studied, what additional information about this pathway were you hoping to gain from your screen?

Interviewee – Ben MajorWell, when we began this study, a comprehensive analysis of the Wnt pathway hadn’t been reported in a human cell line. So, that was one of the major incentives that led us down this line of work. And then, secondarily to that, was to look in a cancer setting where obviously Wnts play a significant role.

Interviewer – Annalisa VanHookWhat about the particular screen that you designed makes it different from previous screens that were done to identify Wnt pathway components?

Interviewee – Ben MajorSo, I guess, very generally, there’s two things that sets this screen off from other screens; the first of which is the degree to which we validated the screen hits. So, this was done in collaboration with Rosetta, and the screen included three phases—primary, secondary, and tertiary screens—and the tertiary screen is what really sets it apart. And that is where we looked at 18 endogenous Wnt/β-catenin and target genes as a readout, across 80 siRNA screen hits. And then, the second thing that makes this screen novel in its approach is that we integrated the siRNA screen hits with a protein-protein interaction network, which again goes to validation.

Interviewer – Annalisa VanHookEssentially what you’ve done, then, is combine a functional screen with protein interaction data?

Interviewee – Ben MajorExactly. Which ultimately yielded a combined physical/functional map of the signal transduction pathway in human colon cancer cells.

Interviewer – Annalisa VanHookCould you give us an overview of the way that this screen worked?

Interviewee – Ben MajorSure. The strategy was very simple. We took colon cancer cell lines, and we engineered them to express a reporter for the Wnt pathway. We then introduced synthetic RNAs, siRNAs, into those cells, we waited three days, and we asked whether or not the Wnt pathway was turned on or turned off. From the hits on that primary screen, we then synthesized new siRNAs and screened those, which, in effect, is validating, through multiple different siRNAs, the hits. And then, the tertiary screen: we took the siRNAs that hit in the primary and secondary screens, and now, instead of using a reporter, we used endogenous Wnt/β-catenin target genes as the readout, and that, together that comprised the siRNA half of our screen.

Interviewer – Annalisa VanHookThen, did you use proteomics data to essentially validate the RNAi data?

Interviewee – Ben MajorYeah, in part, the proteomic network that we both generated from known interactions in the literature, as well as mass spectrometry-based data that we generated, together that created a protein-protein interaction network. And, that yielded four important attributes. So, one, it helped us to validate the siRNA screen hits. Two, it allowed us to demonstrate specificity of our siRNA screen to the Wnt pathway. Third, it demonstrated that proteins that were encoded by the siRNA screen hits formed complexes, as we had expected, but had not shown previously. And then, perhaps most importantly, the proteomics provided mechanistic insight into what the siRNA screen hits were doing.

Interviewer – Annalisa VanHookFrom the screen, what sort of new genes did you identify, that play a role in Wnt signaling?

Interviewee – Ben MajorWe were, in fact, surprised at both the number of new genes that we identified and the majority of these new genes that we identified were, in fact, new and previously unknown. But, when you step back and look at how we performed this screen, it, in fact, makes sense. And, let me expand upon that idea for a second. So, the screen started on day 1, and then 3 days later we harvested the cells and analyzed for activation of Wnt/β-catenin signaling. So, that in fact, allowed 3 days for the cells to change or differentiate or have effects compounding on effects compounding on effects, which at the end of the day, says that the 119 screen hits that we are reporting are not necessarily direct regulators of Wnt signal transduction, but more often than not, they’re going to be indirect regulators. But, to make one last point on this, I don’t really think we care whether or not they’re direct or indirect, if your concern is to better understand Wnt signaling in the context of colon cancer and ultimately with the hope of identifying new therapeutic targets.

Interviewer – Annalisa VanHookWhat did you do to determine how these genes that you identified in this screen contribute to signaling?

Interviewee – Ben MajorWell, as we talked about earlier, the screen itself is incredibly well validated, so the fact that our tertiary screen looked at endogenous Wnt/β-catenin targets, validated that this screen hit was real. The proteomics provided us with some insight into where the target protein was functioning, meaning was it in the nucleus, was it in the cytoplasm, was it associating with a known complex of known biochemical function?

Interviewer – Annalisa VanHookSo, what do you gain from combining functional analysis, that is the RNAi screen, with protein interaction data?

Interviewee – Ben MajorThrough data integration of the two screens, we were able to show specificity of our siRNA screen hits to the Wnt pathway—meaning that if we took 300 random proteins and asked, “Did they hit in our siRNA screen?”, we found that the answer was “No.” But, if we took 300 proteins that are either known components of the Wnt pathway or are interactors with those known components, our siRNA screen hit, did statistically hit, the Wnt pathway. Another thing that the data integration allowed us to see was that the proteins encoded by the siRNA screen hits formed complexes. So, this is expected because proteins do not act in isolation, but rather act in complexes. So we found many orphan complexes, or complexes of proteins that had not previously been tied to Wnt signaling that hit in our screen. We also see hints into crosstalk between the Wnt pathway and parallel signaling cascades. For example, we see that there are proteins within the map kinase-signaling cascade that hit in our screen and functionally interact with the Wnt pathway. So, these kind of insights allow you then to go back to the bench and study Wnt-MAPK crosstalk in the cancer setting.

Interviewer – Annalisa VanHookCan you apply this integrative approach to other signaling pathways, or other cellular contexts? What’s the next step?

Interviewee – Ben MajorUndoubtedly, that’s something that I personally want to do, and Randy’s lab will continue to do. Integrating the two for other signaling pathways is important and will undoubtedly, as I think we have shown, elicit new or provide new disease-related genes.

Interviewer – Annalisa VanHookCould you apply this same sort of screening approach to a different cell type or to a different biological context?

Interviewee – Ben MajorDefinitely. So, we applied the integrative approach to colon cancer cells, but there’s no reason why we can’t, or why someone shouldn’t, apply it to kidney cells or lung cells—because undoubtedly, and I think that the data is out there and is convincing, that Wnt signaling differs between cell types.

Interviewer – Annalisa VanHookDr. Major, thank you for talking with me.

Interviewee – Ben MajorNo problem, glad to do it.

Host – Annalisa VanHookBen Major is lead author and Randy Moon is senior author on a paper published in the November 11th issue of Science Signaling.

That wraps up this Science Signaling Podcast. If you have any questions or suggestions, please write to us at sciencesignalingeditors@aaas.org. This show is a production of Science Signaling and of AAAS, the Science Society. Our producer is Robert Frederick. I'm Annalisa VanHook, and on behalf of Science Signaling and its publisher, the American Association for the Advancement of Science, thanks for joining us

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