IMPaCT Investigator Highlights (Text Version) - Dr. Phillip Febbo

Title: Increased P38 MAPK Is Responsible for Docetaxel Resistance in Prostate Cancer

Investigator: Phillip Febbo, MD, University of California, San Franscisco, Mt. Zion Medical Center

This work is from a Physician Research Training Award that focuses on Docetaxel treatment in men and ways that we can predict who is going to respond to Docetaxel treatment and what are the mechanisms associated with resistance to Docetaxel. Docetaxel is the front line therapy for castration-resistant prostate cancer and it improves survival, yet 60% of men are progressing almost before they complete therapy. And by understanding the mechanisms by which those tumors become resistant to our proven therapy, it gives us an opportunity to figure out combinations of therapies or novel therapies that can prolong their survival.

This project is focused on those men who are in the most dire need of help. We’re talking about locally aggressive prostate cancer, high Gleason—Gleason 8, Gleason 9, prostate cancers that are locally invasive. We ran a trial where we gave men Docetaxel prior to surgery and then we collected those samples at the time of surgery and performed a microarray analysis so that we could ask what pathways are increased relative to aggressive tumors that were not treated with Docetaxel. When we did this analysis we got a list of pathways that looked interesting but it’s always hard to prioritize. So the way we decided to prioritize is we took cell lines and did the same thing. We treated them with Docetaxel and created resistant cell lines and we asked the same question what are the pathways that are up-regulated. And then we put the results together. And what we found is both in the patient tumors and the cell lines that this pathway P38 map kinase seemed to be—have increased expression associated with resistance in Docetaxel.

P38 is a kinase that—that activates through phosphorylation multiple other kinases. And what’s quite interesting is that of the host of proteins P38 MAP kinase can phosphorylate, many of them are what are called microtubule-associated proteins or MAPs. And the reason that’s so interesting is Docetaxel is a microtubule toxin; it targets the microtubules. So it made biologic sense to us that here’s a pathway that could be affecting microtubule dynamics being up-regulated in the setting of resistance to a microtubule targeting agent. So that’s why we really wanted to drill down.

Microtubules serve many functions; they’re critical for the structure of the cell for cell movement and for cell division. And the best way to see microtubules is to label them with different antibodies and you can label acetylated microtubules to see very lacy networks. When we looked at the parental D145 cells versus the resistant cells in the setting of Docetaxel, what we noticed was that the parental cells had multiple abnormal mitotic figures. So these are cells that are trying to divide but there’s been a wrench thrown into the works so they’re not able to divide. And we saw that almost 40% of mitotic figures seen were abnormal whereas the resistant cells under the same concentration of Docetaxel had a much lower percent—less than 20% were abnormal. So if taken at a snapshot we know that given the same exposure, these resistant cells were now having less of impact on microtubule toxicity with the Docetaxel. And the question becomes why.

So one of fascinating parts of this project, and one of the major challenges to understanding microtubule dynamics is how do you measure dynamics. In biology we’re often relegated to taking snapshots of time and comparing different snapshots whereas microtubules are moving in real time and it’s very hard to measure that. Well through a collaboration with Torsten Wittmann at UCSF, we have a method where we can take time lapsed microscopy and visualize microtubule dynamics in real time.

We’ve labeled the protein EB1, End Binding Protein 1, with green fluorescent protein, GFP, and we have a camera that’s sensitive to the GFP. EB1 will sit on the tip of a microtubule as it elongates and that’s why you get these one-direction comets throughout the cell.

Without exposure to Docetaxel the dynamics are quite similar with the microtubules. But once you expose both of these cells to Docetaxel you get a different behavior. The cells round up a little bit and that’s why it’s a little cloudier. But no longer do you see these beautiful comets. You see punctate times when the EB1 is stuck on the end but it doesn’t elongate. It’s stabilized. The resistant cells however are still rounded up a little bit but you do see the beautiful comets. The comets look at little shortened still, they seem to be affected somewhat, but not nearly as much as the parental cell that is not resistant.

We have computer programs that can take these images and quantify the number of EB1 positive comets, the size of the comets, how far they go, how long they go, the speed at which they go. And so this is going to be a platform where we assess the impact of different pathways on microtubule dynamics with or without Docetaxel.

So one of the fortuitous aspects of identifying P38 map kinase as being associated with Docetaxel resistance is that there are drugs that inhibit P38 kinase that have been developed for clinical use. They’re not being used in oncology because as single agents they had no efficacy. But they’ve never been tested in association with Docetaxel treatment. And our research would suggest when we use those inhibitors we don’t see any activity when they’re used alone in these settings. But when we combine them with Docetaxel, we get synergy meaning one plus one equals five.

Across three of our resistant cell lines, a strong synergy between inhibition of P38 map kinase and Docetaxel when used together. So this could be translated and we’re working towards moving toward clinical trials that combine the two.