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A Fifty-Year-Old Cancer Drug Doesn’t Do What You Think

5-fluorouracil (5-FU) has been around a long time now (over fifty years), and it’s a standard oncology drug (particularly in colorectal treatment regimes). But try going around and asking people how it works. If you’re talking to a clinician and want to seem up on the lingo, just say “What’s 5-FU’s MOA?” (mechanism of action).

If your interviewees are honest, many of them will say “Actually, I don’t know”. The others will likely tell you that it’s via inhibition of thymidylate synthase, interruption of which leads to DNA damage. There’s no doubt that 5-FU is indeed a thymidylate synthease inhibitor, and there’s no doubt that that can mess up DNA synthesis, but is that all that’s going on? After all, this is a pretty small molecule that resembles a very important cellular building block. It’s long been suspected that there are other effects.

This new paper actually sheds some light on this topic, and people should prepare for some surprises. There have been many studies before on 5-FU’s cellular effects, of course, but many of these have been at the protein expression level. That’s valuable, but it can miss a lot. Not everything that changes gene transcription is part of a drug’s actual mechanism, because a lot of drug mechanisms don’t go through changes in protein expression. Rather, they set off changes in protein-protein interactions, directly or indirectly. Expression profiles can be sideshows, things that happen because of the main mechanism. Those readouts can be well downstream, mechanistically, and can take much longer to come on than the actual MOA. You can get valuable hints about what might be going on, for sure, but you can also be misled. Taken to an extreme, one risks concluding that piles of wet smoldering ashes are the main thing that causes damage when a house catches on fire.

The authors (a multinational team from Sweden, Singapore, Germany, and England) used the CETSA technique to try to get a more direct mechanistic readout. I’ve written about this a few times here, but the basic idea is that when a protein interacts with a binding partner, it gets slightly more stable. And you can actually see this by heating it up gently and seeing when it starts to unfold – in a bound state, it will do so at a higher temperature than it does in the unbound state. This can be done in vitro by adding a fluorescent dye that reads out on the unfolded state (the DSF assay), and there are other techniques for this “thermal shift” assay as well. What became apparent a few years ago was that you could do this in living cells as well, which opens up a way to get a lot of interesting information that’s otherwise not easy to obtain. This paper uses the latest version of this cellular thermal shift assay (CETSA), which uses protein digestion and mass spectrometry as a readout, and it’s a good advertisement for its power.

The study wisely decided to start by using the active metabolites of 5-FU (5-fluorouridine, FUR, and 5-fluorodeoxyuridine, FUDR) to get the most immediate effects, exposing the cells for two hours before doing the CETSA heating and mass spec data collection. They found several proteins that had already been noted as affected by the drug, which is a good reality check, but they found many more that had never surfaced at all. Interestingly, both FUR and FUDR showed dozens of proteins that read out differently in the thermal shift assay, but there were only seven common hits between the two of them!

As you can see from the names, it’s quite likely that FUR would be going down RNA pathways, among other things, while FUDR would be acting through DNA ones. Indeed, the proteins on the latter one’s list were biased towards nucleoside processing pathways and DNA repair, which suggests that the thymidylate synthase pathway is probably being affected, and that the fluorodeoxyuridine itself may well be getting incorporated into newly synthesized DNA, which quickly attracts repair enzymes when things then gum up.

Meanwhile, the FUR list were largely RNA-handling proteins. These include a whole family of pseudouridine synthase enzymes, two of which had been shown in earlier studies to be affected in nematodes and other species on 5-FU treatment, so that’s a good thing to have picked up again. In the same category is TRMT2A, an enzyme which is believed from other work to form a covalent adduct with transfer RNAs bearing fluorouridine on them, and which has been implicated in 5-FU toxicity. There were also enzymes involved in dihydrouridine synthesis, and there’s a common feature in all of these categories: all of them work on the 5-position of the heterocyclic ring. That suggests that FUR might well be forming covalent adducts in the active sites of many of these, thus leading to a strong thermal shift readout.

It needs to be mentioned that those two-hour cell experiments mentioned earlier involved virtually no changes in protein expression levels (it’s too short a time to see very much of that). It’s all protein-protein interactions that drive these effects – you’d never pick these up by looking at expression levels, and indeed, no one had.

When the authors tried treating an actual colon cancer cell line (HCT15) with plain 5-FU for 12 hours (to simulate real-world conditions), it turns out that the response to the drug seems to be mostly in the FUR-affected proteins. That is, the biggest response to 5-FU treatment is in RNA pathways, not the DNA ones that would be suggested by the putative mechanism of inhibiting thymidylate synthase. In fact, a whole group of tRNA ligases showed up only under this sort of 5-FU treatment (as opposed to using either of its active metabolites). A further experiment used HCT15 cells that had been exposed to enough 5-FU over time to become resistant to it. This experiment showed that the thymidylate synthase pathway seemed to be largely unaffected in the CETSA results, while various RNA-handling protein hits were now changed. Other proteins that showed strong changes in the resistant cells were enzymes like UMP synthase, which are themselves involved in activating 5-FU, so seeing those moving out of the picture is just what you’d expect in a resistance environment.

Taken together, all this suggests that we have been mistaken about the real mechanism of 5-FU. While it does indeed inhibit thymidylate synthase, the more important cellular effects are on the RNA end of things. The authors propose a short list of proteins that could be used in analyzing biopsy samples to predict 5-FU sensitivity (or conversely, to indicate that the drug might be unlikely to work).

This is why I like chemical biology. Opening the hood on mechanisms like this is not easy work, but it’s essential if we’re ever going to put our therapies on a more rational basis. As it stands, we give 5-FU to patients with a mistaken idea of what it’s doing, and when it stops working we really don’t know why. Work like this (and the many other studies that have tried to unravel 5-FU mechanisms in the past) is the only way to change that.

16 comments on “A Fifty-Year-Old Cancer Drug Doesn’t Do What You Think”

  1. David E. Young, MD says:

    5-Fluoruracil has had a glorious history, as any oncologist would tell you. I can remember the intense debate back in the late 80’s and early 90’s about what dose of Folinic acid (Leucovorin) was optimal for gently improving the safety and efficacy of 5-FU. There were these complex diagrams showing the various pathways the 5-FU took. We also learned that bolus 5-FU was a different animal than infusional 5-FU. The current optimal treatment for colon cancer uses both bolus and infusional 5-FU as if they were different drugs. (code name FOLFOX treatment: Fluorouracil two different ways, Oxaliplatin and Leucovorin). There was a time that some of us added Trimetrexate to the mix, but the data was weak and ultimately Trimetrexate went the way of the Dodo bird. Curiously, although everyone agrees that combining Leucovorin with Fluorouracil improves effectiveness and safety, Leucovorin is considered incompatible with Fluorouracil in every EHR program. As an oncologist, I have to sign off that I understand that Leucovorin makes Fluorouracil worse when ever I order Folfox treatment. Fluorouracil. the worlds most complicated simple medicine.

    1. Mike Owens says:

      Dr. Young, only proper nouns are capitalized within a sentence. You post here often so I hope you, and others, find this useful.

      1. Jose says:

        Although i am aware of the expectations in a journal article, i found the germanic capitalizations helpful in reading the post, even if you found them distracting.

        1. Mike says:

          Jose, it’s just plain wrong. You don’t capitalize water or oxygen. Indeed, it confuses readers who assume that these may be brand or trademark names.

          1. Anonymous says:

            Umm, they are brand names.

        2. sgcox says:

          Mike is of course absolutely right.
          It is Humira but adalimumab and Tylenol but paracetamol.
          However, Germanic capitalizations used by Dr. Young makes article so much easier to read and focus on the message. Even if it is not right, I like it.
          Also, adalimumab is a very specific and very well defined entity, not hydrogen or oxygen and I think it fully deserves capitalization., may be even more than Humira.

  2. Francois says:

    The mode of action of many (most?) approved drugs is unknown…

    1. FoodScientist says:

      Unless it’s Aducanumab. Where the mechanism of action is known and the efficacy unlikely.
      The protein fibrils seem more like the wet smoldering ashes to me.

      Targeting some of the kinases/phosphatases is the logical approach. The phosphorylation sites are what controls the protein stability. It’s a tougher target(s) because there are multiple different enzymes that have preferred sites they act on. They also do a lot of general other stuff, so the side effects could be unpredictable.

  3. J says:

    Now that we know the active metabolite, it’s only a matter of time before it gets patented. I propose the trade name “Adrunex.”

  4. Tom A says:

    That’s “wayback” territory for me. I remember some researchers working with 5-FU and other analogues when I was briefly at Roswell Park Institute back in 1967. I was conscripted to work proline analogues, like pyrazolidine-3-carboxylic acid. My fondest memory is using liquid bromine. Good times.

  5. David Edwards says:

    “Opening the hood on mechanisms like this is not easy work”

    Neither is making the requisite research accessible to a non-technical audience.

    This, and numerous other of your articles, are the reasons I keep coming back, and not just for the hilarity of TIWWW. You have real skill in this department, and it’s not only the TIWWW posts that deserve a book, but several of these posts as well.

    This post is also an object lesson in how science works – not only by finding new entities and interactions, but discovering that entities considered familiar have hitherto unexpected aspects thereto. Indeed, learning how to understand the purportedly familiar more deeply pays dividends as here.

    Now it’s time to launch the paper hunt …

  6. Adam Zweifach says:

    Hmmm… maybe write a piece about the drugs that actually *do* work the way you think they do. It’ll be short.

  7. David Taylor, MD, PhD says:

    Good essay — it reminded me of the news, only a few years ago, that the therapeutic mechanisms of aspirin had finally been discovered.

  8. Dennis says:

    After doxorubicin, yet another 50 year old anti-cancer drug that does not quite do what we thought it did…

  9. Brussels bureaucrat says:

    Excellent presentation of an interesting study, as usual. However, I am a bit sceptical for two reasons. Some years ago a Nature paper (cant find it right now) provided convincing evidence that most drugs bind more than ten targets and that binding does not equal effect. Secondly, in all mass spectrometry-based proteomics you have to take into account the “dynamic range problem” – proteins are present in very, very different concentrations and proteins involved in RNA processing (including ribosome proteins and associated proteins) are among the most abundant proteins in cells and thus for this reason alone more likely to bind any drug.
    I it always easy to criticise, but I need a bit more mechanistic evidence to be convinced.

  10. Norbert Stumpf says:

    “Problematic” is a polite summary description. The mechanisms of cytotoxicity of 5FU that predominate in a given situation are highly dependent upon concentrations and exposure times, and the concentrations of FUDR and FUR that they used are not even close to clinical exposures to 5FU in common regimens such as FOLFOX. Plasma 5FU concentrations during the 46 hour 5FU infusion in the FOLFOX or FOLFIRI regimens are less than 5 micromolar, far below the 100 micromolar concentrations of the metabolites FUDR or FUR tested in the publication. A simple test of relative roles of RNA-directed versus TS-mediated cytotoxicity is to determine whether supplementation with thymidine versus uridine reduces toxicity and/or antitumor efficacy in a given system. In vivo, antitumor efficacy of tolerated 5FU regimens in both mice and humans is overwhelmingly due to TS inhibition, whereas dose-limiting toxicities of 5FU are largely mediated through 5FU misincorporation into RNA and are reversible with exogenous uridine administered a number of hours after the 5FU, as was demonstrated in the 1980s by Dan Martin, Frits Peters and others. In mice, low doses of FUR (e.g. 2 mg/kg in mice) cause raging toxicity due to incorporation into RNA, with relatively little antitumor efficacy, whereas FUDR is tolerated at doses of several hundred mg/kg. Anyway, such fancy studies on protein interactions should do a much better job of taking clinical pharmacokinetics and basic pharmacology into account if the intention is to eventually improve or guide clinical practice.

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