Last time I told you about how the view of cancer switched from the perspective of metabolism to oncogenes. Today we'll see how recent developments have placed the spotlight back on metabolic pathways.
I'll begin this tale with a quote from a review written by Andrew M Arshama and Thomas P Neufeld:
The TOR (target of rapamycin) signaling pathway has been the subject of a 30-year-long reverse engineering project, beginning in the 1970s when the macrocyclic lactone antifungal compound rapamycin was purified from soil bacteria found on the Pacific island of Rapa Nui, famous for its moai (giant carved heads).
The story begins with this funny compound that was named after the island of Rapa Nui, aka Easter Island. Rapamycin had very interesting properties, it not only acted as an antifungal agent but inhibited the immune system and slowed down cell division in mammalian organisms. In 1991 it was discovered that the target of rapamycin in yeast was a kinase that was then named TOR, for "target of rapamycin". Curiously, TOR was present in mammals, vertebrates, plants ... it was a pretty old protein, and was the central player in an ancient signaling cascade.
A couple of digressions and then we'll move on.
First, it is now known that rapamycin inhibits TOR activity by binding a complex formed between TOR (or mTOR in mammals) and a second protein called FKBP12. Second, kinases are enzymes that can add phosphates to specific locations on other proteins. Often the addition or removal of a phosphate will change the activity of a protein. As mentioned in Part I, kinases often phosphorylate other kinases. When one kinase activates a second kinase which in turn activates a third, this is known as a signaling cascade. Of course other types of proteins can act in such a cascade (G-proteins for example), but usually these "pathways" are full of kinases.
OK back to the story.
While some scientists played with rapamycin, other investigators were asking a different question: what regulates cell size? A very academic sounding question, but one that gave very intriguing answers. When cells duplicate their DNA and accumulate enough cytoplasm, only then do they divide. Cytoplasm is the cellular goo found inside the cell and outside of the nucleus. It's jam-packed with proteins, about 300 mg/ml, and contains everything that the cell needs, including ribosomes, sugars, organelles. It turns out that cells closely monitor their size and quantity of cytoplasm. This assement of size is accomplished by the activity of a bucket full of genes that are thought to form some sensing device. From a few genetic screens, it appeared that some mutant cells didn't wait to make enough cytoplasm before dividing. These cells had normal cytoplasm, just a lot less of it - in other words these mutants were smaller. The fundamental questions became:
1) How do cells sense the total amount of cytoplasmic components (proteins, ribosomes etc.)?
2) How do cells regulate the production and destruction of these cytoplasmic components?
Curiously, it was not only the size of the cells that were affected, but also the size of the animal. For example, two mutant flies that had small cells, also had small body sizes. The mutated genes were named hamartin and tuberin and encoded very conserved genes. Many other signaling molecules were picked up in those genetic screens performed in flies, including genes involved in insulin production and detection. From the insulin result you might be thinking - Aha so cells that are messed up in sensing sugar don't grow well. And indeed, for many years, researchers identified mutants that acted as if they were subjected to starvation in the presence of normal levels of nutrients. To grow properly, cells need amino acids, sugars, phosphates etc. If you starved yeast, they stop making protein and stop dividing. But it wasn't just a case of cells passively taking in nutrients and growing; instead, it seemed like cells had some sophisticated method of measuring the nutrient availability. Based on this nutrient sensor the cells decide whether
a) to grow, make proteins, divide and be happy
b) to stop protein synthesis, conserve energy and turn off cell division
c) and if things were really bad, to make protective proteins, eat its excess organelles and really prepare for the upcoming disaster.
Over the years, researchers found mutants that acted as if they were being starved even while in the midst of plenty. One such mutant identified in yeast was a protein called Rheb. This G-protein was similar in sequence to Ras, a nasty oncogene, and seemed to be required for the cell's ability to sense its metabolic condition. Mutant cells that lacked Rheb were stressed out even when there was no need. They stopped making amino acids, they shut down protein production and started to eat their own organelles (the process of eating yourself is known as autophagy).
So lets sum it up:
- rapamycin: stops cells from growing and dividing
- tuberin hamartin mutants: small cells (non-functioning cell-size sensor?)
- insulin mutants: small cells (non-functioning cell-size sensor? non-functioning metabolic sensor?)
- Rheb mutants: stressed out cells (overactive "lack of nutrient" sensor?)
Then some scientists started to add the facts together and thought that perhaps rapamycin had something to do with this nutrient/cell-size sensor? Sure enough, TOR mutants were paranoid little fellows. As with the Rheb mutants, TOR mutants acted as if Armageddon were upon them while swimming in a sea of milk and honey.
So what's the link between metabolic sensor mutants and size sensor mutants? Biochemists filled in all the connections between the players discovered by the geneticists. It turned out that all these genes were part of an evolutionarily conserved signaling pathway that acted to sense all sorts of metabolic levels. Metablic and cellular function information would be funneled into one master signal integrator, TOR, that would then turn on specific cellular programs. If the signaling pathway was overactive, cells would act as if they were swimming in ample amounts of nutrients, even when they weren't, and would divide before they had produced enough cytoplasm, leading to the small cell/small animal phenotype. If the signaling pathway was dampened, cells would act as if there wasn't enough and turned on all these stress signals. It looked like the TOR signaling pathway was the master metabolic sensory system of the cell.
About the same time as all these pieces of the puzzle fell together, other scientists discovered that quite a few of the nastiest tumor suppressors and proto-oncogenes also affected the activity of the TOR pathway. Some examples are Phospho-inositide-3 kinase (PI3K), Akt and the phosphoinosited phosphotase PTEN, one of the most important tumor suppressors. In addition, when the mammalian homologs of the hamartin and tuberin genes were mutated, the result was ... tuberous sclerosis, a disease associated with the apearance of numerous benign tumors. PI3K itself was regulated by growth factors and their receptors. Now the circle was complete.
A big picture of cell division was coming into focus. Cells need to sense many things before they can begin to divide. They need to take into account whether they have enough metabolites to make more proteins, ribosomes, and all the goodies needed to furnish the two new cells. In addition, the cells of multicellular organisms must work together. A cell can only divide if the larger organism needs more cells. There was no need for the cell to activate the cell division program if there was no need to replace tissue or for the multicellular organism to grow.
So let's draw out a diagram of what we know so far. When one set of proteins activates the next set of proteins, the connection is represented by an arrow. When one set of proteins (or factors) inhibits the next set, the connection is represented by a line ending with a blunt end.
Note that the most mysterious part of this pathway is how metabolites affect the tuberous sclerosis proteins (TSC1 and TSC2).
As you can guess, this signaling pathway is at the heart of what the cell has to do: whether to live a swinger's life full of fun and reproduction, a peaceful life of cellular-retirement, or a stress-filled life of poverty. Signals used by the cell to make this decision come from both metabolic sensors and cell-to-cell communication sensors. If a mutation arises in a gene on any of these branch points, you could wind up with an over-stimulated cell that divides way too fast (i.e. cancer).
As you can see we've come full circle. Cancer was a problem with signals - those that sensed metabolites or those that sensed cell-to-cell communication.
Some further notes: Judging from this diagram, you might think that Rapamycin, which inhibits TOR activity, must be a fabulous chemotherapeutic agent. But it isn't. Why? Well it turns out that there is a major feedback loop in this pathway. Basically TOR inhibition activates Akt. You don't want to mess with Akt: it's a major oncogene and its activation regulates many pathways that promote cell division.
Recently a lot of investigators have looked downstream of TOR to see how exactly it stimulates cell growth, in terms of how big cells get and in terms of how fast cells divide. And in an interesting twist, it seems as if TOR's main purpose is to regulate how the ribosome operates. First off, TOR signaling turns on the expression of ribosome proteins and ribosomal RNA. Since the ribosome is the main factory in the cell, it is no surprise that a regulator of cell size would also regulate ribosome production. But that's not all. Active TOR actually sits on ribosomes as they get ready to translate an mRNA into protein. This active TOR adds tons of phosphates directly to ribosomal proteins and even activates other kinases, such as S6 kinase, an enzyme that adds even more phosphates to the ribosome (S6 is a small ribosomal subunit). Yes, TOR activates a big phosphorylation orgy on the ribosome; however, it remains unclear what is the outcome of all these ribosomal phosphorylation events.
In addition, TOR signaling promotes the ability of RNA unwinding proteins (aka helicases) to remove RNA folds from the start of certain transcripts. TOR accomplishes this task through eIF4E, a protein that I've written about before. eIF4E is a regulator mRNA translation. It binds to the cap present at the beginning of most transcripts and can recruit helicases (eIF4A and eIF4B). In the absence of TOR signaling, the 4E inhibitors (called 4EBPs) prevent eIF4E from recruiting the helicases to the start of the transcript. When TOR is active, it phosphorylates eIF4E inhibitors and turns them off. eIF4E recruits helicases and the ribosome can now translate the mRNA in question. Having told you all this, I will now say that TOR signaling normally doesn't affect the translation of most mRNAs, but it does have a profound effect on the translation of a small set of transcripts that contain extensive RNA folds right after the cap. And what are these transcripts? Here is the real kicker. Many are mRNAs from proto-oncogenes whose protein products are critical for promoting cell division. From very recent experiments in Nahum Sonenberg's lab, we found out that just increasing the level of eIF4E by two fold can lead to the over-expression of a whole group of proto-oncogenes like myc. In fact, many drugs that target eIF4E are in clinical trials for a variety of cancers. So in the end it looks like the cell's metabolic state could stimulate the translation of genes that promote cell division. How's that for a turn of events?
In the next entry I'll talk about some of the newest TOR signaling papers (some published, and others soon to be published), and a surprisingly new connection between metabolism and cancer.
-I've been furiously typing this post in between experiments - I'll be correcting any typos over time. I'll also try to stick in some references here and in the previous entry as soon as I can get to it. Also feel free to add any extra info in the comments section. I didn't want to complicate this entry so note that I did not talk about TORC1 & TORC2 or autophagy, or memory or any of the gadzillion processes affected by TOR signaling.
I have nothing to add rather than saying that this is really fantastic summary of the TOR story. Thanks for posting it, looking forward to your next entry.
Great blog, thanks! Clear, interesting/appropriate details, nice style. Added to my regular rounds.
(I was pointed here from ouroboros.wordpress.com.)
I am trying to make mTOR signalling understandeable for my non-biologist co-workers and your blog has helped me immensely - thanks! The pitfall of AKT upregulation at mTOR inhibition is substantial, but can be reduced if mentioned, that rapamycin (or analogs) cannot block the action of TORC2, which is the activator of AKT. Unfortunately at permanent addition of rapamycin, it can inhibit the formation of new TORC2, which may lead in consequence to the feedback loop you have mentioned - but at the late stage of the cancer when the drugs are administered, this might be your smallest concern...
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