Thursday, April 14, 2016

Elegant study on KRAS shows how valuable proteomics really can be in studying diseases.

Don't watch this video yet!  But it is fantastic.

KRAS is something that researchers (including a bunch of my friends!) have been tirelessly working on for 60 years or so. Why? Cause its crazy important. 95% of pancreatic cancers have KRAS mutations. And we all know how pancreatic cancer almost always turns out.

One of the first things Harold Varmus did when the POTUS appointed him to helm the NCI was set up a focused RAS Initiative project to get to the bottom of this stuff. And a ton of smart people are working hard on RAS from funding from this initiative and many others. We've got to figure this stuff out. And, because it is a mutation, well -- most of the work has been done via genomics and transcriptomics. Makes sense, right? Proteomics hasn't been ignored, but you could argue pretty easily it certainly hasn't been the focus.

Ben -- take a deep breath. While I'm building a wall of self-control around my enthusiasm, I suggest you check out this paper from Chris Tape et al., that came out in today's CELL (and is open access).

One super dangerous thing that we got into in early cancer work was studying cancer cells in isolation. We learned a lot by taking cancer cells and getting a pure growing culture of the immortal cells. That's how you'd do it in microbiology, and cancer was studied the same way. The problem is that cancer isn't an isolated event. Sure, there are bunch of gross cells growing all crazy, by themselves, but they have to interact with all the cells around them.

SO, this is where this super smart study comes in. I don't have my head fully wrapped around it (its a CELL paper, after all). They did the normal stuff, of course. Did some quantitative proteomics of their interesting KRAS cell lines, quantified some cytokines and verified previous findings about growth factors. Nice start!

Next? Well, they take this KRAS cell line and do thorough quantitative phosphoproteomics (SILAC based) on the cell line with various inhibitors of phosphorylation that are important throughout the pathway in the cell. RAS pathways involve all sorts of key operators JAK/STAT, ERK, heck, you name it. So...they inhibitied distinct pathways -- to determine how KRAS phosphorylations proceed under these conditions. Stop there, you've got a nice paper!   Nope, we haven't even gotten to my favorite part.

Okay, I'm going to even skip the point where they do TMT 10-plex for mult-axis phosphoproteomics (WHAT!?!?!) cause I've got a lot of work to still do tonight.

Lets go back to thinking about cells in their native state. Not all cells go bad. Some cells go crazy and eventually divide out of control. But they are surrounded intrinsically by good cells. So. What if you wanted to study this? Would you take some of your whacko cells and grow them up and mix them with good cells and see what happens? Maybe, but how would you tell what phospho cascades are changing in what cells?

You SILAC label the two different populations differently.  And that is what they did. You take all the cells and then do phosphoproteomics on them and you can see that the KRAS cells are secreting signals that cause the stromal (surrounding) cells to come to their aid!

Something like this --

We've known for a while that stroma plays some role in the development of pancreatic cancers, but an awful lot of sequencing has turned up very little. Its protein level post translational modifications that are to blame here. These beautiful, natural, phospho signaling cascades are being diverted -- not only within one cell, but to affect cells that are technically not cancer cells on their own. Will this have some affect at the genetic level? Sure, eventually, as these signals lead to differential regulation of transcripts. But we're missing an awful lot of the picture here and I bet a lot of observations of real tumors are going to make a lot of sense now in this context.

I could go on about the validation, but, again, I've got a long night ahead.

Absolutely stunning paper.

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