Chapter 13: A Twenty-Year Study that Promises a Paradigm Shift: The Yeast Two-Hybrid System

Chapter 13: A Twenty-Year Study that Promises a Paradigm Shift: The Yeast Two-Hybrid System

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In this chapter, Dr. Hung describes his involvement in controversial research on the Yeast Two-Hybrid System. He sets the context by explaining the prevailing theory about how cell receptors interact with proteins, noting unexpected discoveries linking a receptor to activity inside a cell nucleus. Dr. Hung has built on this discover and traces the history of publishing his findings. He speculates on why they have not been accepted. He explains that he has always been convinced that there is something significant in this finding and notes that his laboratory has continued to work on the mechanisms of how the signals move from the surface receptor to the nucleus of a cell. His laboratory has demonstrated that these signals influence DNA repair and transcription and therefore have implications for anti-cancer therapy. He notes that his laboratory has been able to link the Yeast Two-Hybrid system to functions involved in liver regeneration. In the remainder of this chapter, Dr. Hung makes general statements about how basic scientist must focus on the reproducibility of data, rather than accepted dogma, to guide the discovery process.

Identifier

HumgMC_03_20140421_C13

Publication Date

4-21-2014

City

Houston, Texas

Topics Covered

The Interview Subject's Story - The Researcher; The Researcher; Character, Values, Beliefs, Talents; Overview; Definitions, Explanations, Translations; Discovery, Creativity and Innovation; The Professional at Work; On Research and Researchers; Understanding Cancer, the History of Science, Cancer Research

Transcript

T.A. Rosolowski, PhD:

Yeah, yeah. Incredible, incredible. Where would you like to go next? I know that there was….

Mien-Chie Hung, PhD:

Sure, okay…

T.A. Rosolowski, PhD:

… do you want to talk about the research …

Mien-Chie Hung, PhD:

Sure.

T.A. Rosolowski, PhD:

… and since we’re kind of on the idea of …

Mien-Chie Hung, PhD:

Yeah.

T.A. Rosolowski, PhD:

… paradigm shifts.

Mien-Chie Hung, PhD:

The paradigm shift. These one was in early 1990. I was interested at that time that not much was known about signal pathways for this growth factor receptor including ER receptor and HER2/neu. They are also called growth factor receptor and by definition, they’re receptors --- receptor meaning they were outside of this --- outside --- connected outside, inside the cell, right? And then you have a ligand ____ 19:43. At that time, our knowledge was that when you have ligand from outside the interactive receptor, then receptor worked just like a --- a relay, passed the knowledge to A; A pass to B; B passed to C, and all the way toward this, transferring the signal from outside of the cell, going to nucleus to cells, to tell the cells, hey, now you should replicate. One becomes two, the DNA has to replicate, then one cell become two, two becomes four, four becomes eight, and so on, right? So that’s our traditional concept back to that time. So the receptor should be in the cell surface but not ___ 20:20 in the receptor. We were very surprised by an experiment that we were doing with a so-called yeast 2 hybrid system. That is, trying to identify a cellular protein –using a special technique and trying to identify what cellular protein may interact with the receptor, their --- their cytoplasm portion. Because the receptor has, you know, a certain memory, right? One part is outside, the other one part is inside. So they go. So we are interested to identify this A, B, C, the so-called, now the so-called signal. And there’s a special technique called Yeast 2 hybrid system.

T.A. Rosolowski, PhD:

What is that it?

Mien-Chie Hung, PhD:

It’s called yeast …

T.A. Rosolowski, PhD:

Yeast?

Mien-Chie Hung, PhD:

East 2 hybrid system. That’s where the basic scientist….

T.A. Rosolowski, PhD:

Yeast 2….

Mien-Chie Hung, PhD:

2 hybrid…

T.A. Rosolowski, PhD:

.. hybrid. Oh, got you.

Mien-Chie Hung, PhD:

2 hybrid system. But that’s a special technique. But from that study, we actually had an unexpected discovery. HER2/neu associates with transcription RTK And, as you know, transcription occurs in the nucleus and HER2/neu within cell surface. It --- it’s a receptor. But how can that receptor have transcription RTK? Just like the Chinese are supposed to be in Beijing, but now you see the Chinese, you know, two or three hundred you see a Chinese in New York speak English. It is just --- just impossible, right. So, but the data is reproduceable 21:49. So, i.e. we discovered a very unexpected result. A cell surface receptor could associate with the transcription RTK and apparently occur in the nucleus. And this is certainly anti____. This certainly not a subject and fortunately the experiment is reproducible. What’s important is the cure. The experiment had to be reproducible. So when experiment reproducible, then I start to feel as if the experiment is reproducible, there are two things. First, they are probably real and important.

T.A. Rosolowski, PhD:

They are probably …

Mien-Chie Hung, PhD:

Real …

T.A. Rosolowski, PhD:

Right.

Mien-Chie Hung, PhD:

… and important.

T.A. Rosolowski, PhD:

Yeah.

Mien-Chie Hung, PhD:

But people had just not discovered that. Second --- yeah, they are reproducible but they may not be important, which is less likely. The cell will not just have something happen and it’s without some meaning. However, nobody believed this concept. So we discovered that --and my student, ____ ____, that he’s a professional in Winston 22:57 University now. At that time, it’s in early 1990 that we could not figure out what --- what is ___ relevant. We thought it’s very important to start, so we went and wrote a paper for Nature and Science, you know, those are major journals. Of course, it been reviewed because it very novel. But it has not been accepted. And now, back --- I look back, I think it’s fair because we made a very novel discovery but we did not say why physiologically that’s important. And that may be a very interesting discovery but this may be nothing to do with the biology, right?

T.A. Rosolowski, PhD:

Right.

Mien-Chie Hung, PhD:

But then after that, we published a very small paper in BBRC. It’s a very fast communication journal. Luckily, we published it, given it’s a small journal. Because it’s now on record, we are the first to report that --- that the receptor associated with transcription RTP is in the nucleus. We are the first. If we didn’t publish it, we would never have a record --- we would never have a record. But then, after that, it is difficult field to study. Paper was very difficult to publish and technique is not --- I’m not --- the --- the paper difficult to publish. I’m not complaining anything, I am just saying because of the technology at that time, it’s not easy to convince people, say that’s real. It’s difficult, you know, technical-wise. So I always try to ….

T.A. Rosolowski, PhD:

So, excuse me … just … because of the technology involved, was the assumption that there are --- might have been some kind of, you know, error in the technology …

Mien-Chie Hung, PhD:

Yeah, yeah, yeah. ___ …

T.A. Rosolowski, PhD:

Okay.

Mien-Chie Hung, PhD:

___ For example --- for example, I say nuclear. I can take a cell and break the cell, isolate the nucleus and say, hey, it’s there. But technically, it’s very difficult to say whether you have small amount of contamination.

T.A. Rosolowski, PhD:

Right, okay.

Mien-Chie Hung, PhD:

Right?

T.A. Rosolowski, PhD:

Right.

Mien-Chie Hung, PhD:

So unless you see directly. But now there are more and more data now. Even more recently now, you can see a single molecule by trafficking, you know, after twenty years. The technique has improved. At that time, the experiment was a very difficult experiment to do, to show. But I --- since the data is reproducible, I firmly believe something is there. So therefore, from HER2, originally we discover HER2, then we switched to EGF receptor. And there is another reason to switch to the EGF receptor. One is not only HER2, but also EGF receptor is a problem, right? So if --- and this was done by different person so I’m more convinced that’s real.

T.A. Rosolowski, PhD:

Interesting.

Mien-Chie Hung, PhD:

Second, HER2 has no ligand. HER2 up to now is still --- is an orphan receptor because there is no ….

T.A. Rosolowski, PhD:

Orphan receptor …

Mien-Chie Hung, PhD:

There is no ligand. So for research per se, the EGF receptor is easier because of your ligand stimulation 25:45. So then, these two are very, very close. At that time back to the old day, we almost did these two side by side, because it is a very unusual phenotype. So only unless we have two different people, one seeing HER2 and one seeing the EGF receptor am I convinced this is real. So the first paper was published in 1994 in BBRC, although our observation in 1991 we already sent to Nature. The paper couldn’t accept it because --- which --- which is fair, which is fair. I’m not complaining. Is, you know, scientifically, when is come out, is novel idea, a very unusual stretch. You have to show enough, otherwise you are --- you are generating junk. So our second paper on this area is 2001 …

T.A. Rosolowski, PhD:

Wow! That’s a big gap.

Mien-Chie Hung, PhD:

… in Nature Cell Biology. And it doesn’t mean that for seven years, I lie on the beach, okay? That same year, I was working with something else, trying to get funding to do it but using other funding to support this project and continue. And try to convince people to do it. People don’t want to do it. Because student ____, it’s difficult to publish and difficult to do. But I believe it’s real. Because multiple people who did it until 2001, it was a more --- in a more well known journal, now it’s in Nature Cell Biology, 2001. After that, people started to gradually believe it. And then, we started to back to literature. I thought we had first one. In HER2/neu, we are the first one to --- we are the first one to identify the transcription activity, but in terms of receptor in the nucleus, even before us, we are first one to discover HER2. Someone else already showed the EGF receptor in the nucleus.

T.A. Rosolowski, PhD:

How interesting.

Mien-Chie Hung, PhD:

But they don’t know what that means. And then, there are more and more people showing different receptors in the nucleus. But the --- the biological activity is just difficult to study because keep in mind it’s a cell. Also, ___ 27:35 it’s easier to study because you touch it. In the nucleus, the small amount it is always more difficult to study. More difficult to study, but this means as important. It’s just historically you have to start from easy studies because of more difficulty. So we happened to discover. So up to now in the receptor tyrosine kinase, is the cell surface receptor. There are 20 --- more than 20 subfamilies, superfamilies. There are more than 10 --- more than 10, and more than 50% of them had been reported as being detected in the nucleus by all different groups. But the function is not as clear. The function study --- the function study, the one, the major lab for the country can’t do more for this study on this particular area. I think my lab is the major one. And yeah, there are other people, too, but we are the ones who have been consistent in the 20 years. And now we know this receptor tyrosine kinase from cell surface can move into the nucleus. We know the mechanism and how they move in, and we know they’re involved in transcription regulation. We know they’re involved in DNA repair. So if a receptor could actually from cell surface move to nucleus and become involved DNA repair and transcription, this got to be important. And now, there are even more data which are from us and from other people showing that these receptors, when they go to nucleus, actually cause a resistance to certain types of anticancer therapy. And so, I think function will be gradually unraveled. It’s not completely a, you know, shaking-earth impact yet but it’s gradually developing. It’s more than ____ (00:29:20). Now, there are people starting to accept this concept, and that is what’s important to show. And this particular one, I still strongly believe in the near future, this will have to be in textbooks. The reason I’m saying this: because when we talk about signaling, we said cell, now cell biology textbook, is --- in the textbooks they say a ligand stimulates a receptor then the receptor passes through those signaling like a relay; A pass to B, B pass to C, then goes to nucleus. Which is still correct. But in addition to that, there’s another pathway. It’s a memory receptor. They --- they don’t necessarily pass this way by a relay. They --- by itself, in the vesicle it translocates all the way to the nucleus and by itself, directly participates. And the first time, when the EGF receptor was found to be in the nucleus, when they discovered it, it was doing a liver regeneration. Liver regeneration is a kind of experiment that take any more liver. You cut the liver out. The liver is important. When you cut a liver out, the liver has to quickly regenerate. And when it quickly regenerates, using an electron microscope, they see the EGF receptor nucleus. That was not done by us, it was before us. And they didn’t know what it means. My interpretation, it’s an SOS. SOS why? So usually you call these pathways but now because of liver regeneration --you want to quickly regenerate the liver. So why wouldn’t this characteristic do something. So now, there are more and more of these cell surface proteins. At the very beginning, people thought it was cell surface protein, then we said it’s nuclear. But now --I’m not alone in this-- people started finding that these cell surface proteins can go to different compartments of --- in --- you know, inside the cell, there are lot of different compartments – mitochondrial, Golgi, you know, ER, called the nuclear membrane, you got the nucleus. Just, i.e., they’re cel --- intracellular compartments. These so-called cell surface receptors, one by one, have been discovered. We happened to be more focused on nuclear but now, this kind of cell --- so-called cell surface protein, you can actually detect it in mitochondria, in Golgi, in ER, nucleus. And they --- when they locate it different place, they have a different function. There is more and more of this kind of stuff. Which is common sense, okay? Three hundred years ago, you go to Europe, it’s very difficult to find a Chinese. But in a different, you see world, you can see Chinese in France, you can see Chinese in Italy, and Chinese in Germany – just like that. Historically, we didn’t know, we said the cell surface protein is on the cell surface. But cell surface protein can be internal. It can go to different locations and do different things. And if --- if nature --- that’s occurring that way and they have an important function which is ____ 32:23. We --- as a --- as a scientist, is basic --- I’m talking about basic science now, we try to use scientific knowledge to discover nature. If nature --- if that occurs in nature --- when I say occur in nature, meaning cell have that property, something is important there. So --- and then, we have to think about other parts because historically, all of the science was discovered because of people before us, they contribute, they make a discovery. They write a theory. But in reality, even one hundred years from now, we still have not completed all the science yet. How about --- we think about 40 years ago, how much did we know? We knew so much 40 years ago, and 40 years pass, and now we know much more than before, right? And a lot of stuff we discovered at that time, historically you thought that way. So we think that’s --- I --- I --- I always, when I give a lecture on this one, my last slide always states that –that’s an interesting story. That is, seven blind guys try to figure out an elephant. Scientist is like that. We, as scientists, we try to use scientific knowledge to under --- understand nature so we design experiments and then we use our experiments as ___ 33:42 system and that experimental result come out. That’s what we like and thus we start with step by step establish our knowledge. Just like the seven guy. The seven guy --- the seven blind kid touch elephant and when he touch it, the first one he touch it most likely in this area because it’s huge. Then he say, “So what is elephant? It is like a wall,” right?

T.A. Rosolowski, PhD:

It’s growing more and more.

Mien-Chie Hung, PhD:

[34:8.2] Yeah. And he’s --- he’s right. But how about another person, when he touch elephant ear, then you say, “what is an elephant?” “Oh, like a fan,” right? So --- and --- if they were touch it at the same time, they may argue but if they touch at different time, “But this one is much larger.” So many people come to touch it so what is elephant? “The wall.” And the other says, “The wall? It look like – a wall. Then that’s wall.” Then 10 years later, another person touch …

T.A. Rosolowski, PhD:

The trunk, yeah.

Mien-Chie Hung, PhD:

And they say, “It look like what?” “Oh, maybe snake? I am wrong.”

T.A. Rosolowski, PhD:

How could --- how --- how could those other people possibly have said it was a wall…

Mien-Chie Hung, PhD:

It’s wrong. That --- it --- that’s --- it happens all the time. So I start to educate my trainees. As long as data is reproducible, what’s important is get high data reproducibility --- reproduce. The data not ___ 34:57, it all ___ anyway. Data reproduce --- data reproducibility, you have to think about what it means. And you have to think out of box because many, many, many, many things in the textbook --- I’m not saying they’re wrong, they’re right. But they are incomplete and that’s why scientists need to be generation after generation in discovery mode. But if you always say whatever the textbook says is right, then you’re not going to make an important discovery. Right? So but --- but what’s important is a high degree reproducibility. And so this --- I’m still working on it. And I --- now I think we already know the mechanism, how they --- how --- originally don’t even --- cannot even imagine how --- how a protein from cell surface can go to nucleus. We know --- we know the entire mechanism and now we try to design --- incorporate with our bioengineer, try to see if we ca --- can see, using different kind of technology, to see it and show its importance. So --- I --- I firmly believe this will be in the textbook. This is different from cancer now. This may be related to cancer, too, but may not --- not directly related to cancer but similar to the other project which I mentioned, the translation. These ideas are more in basic science but, however, they are still related to cancer.

T.A. Rosolowski, PhD:

But it’s interesting. I mean, you’re --- your focus on that story and, you know, before we turned on the recorder, you --- you used the word “persistence.” I mean, you have an intuition that there’s something going on here …

Mien-Chie Hung, PhD:

Yeah.

T.A. Rosolowski, PhD:

… and so you’re sticking with it. And who knows?

Mien-Chie Hung, PhD:

Yeah.

T.A. Rosolowski, PhD:

I mean, it may even be tangentially connected. And so….

Mien-Chie Hung, PhD:

Yeah.

T.A. Rosolowski, PhD:

… you have to follow the pathway literally.

Mien-Chie Hung, PhD:

And --- and --- and at that time, I told myself since the data is highly reproducible, right, so it’s real. And as scientist, I don’t want to say because the data reproducible and because it --- nobody believed it so we should not do it. And I understand it’s difficult to get results published. I know it’s difficult to get funded. But a lot of important stuff was not funded, including the Cetuximab that was Mendelsohn’s antibody. At the very beginning, he could not get funded. Remember that story?

T.A. Rosolowski, PhD:

[37:1.4] Yeah, yeah.

Mien-Chie Hung, PhD:

But it’s now, it’s an anticancer drug. So as a scientist, I feel if it’s real, you know, I know it. If I know it, it is our responsibility to ___ 37:13 up. And unless the data is nonreproducible, that’s --- if its reproducible, we should do it, we should do it. We should do it. And if it‘s difficult, do it slowly. If you want to do faster, you get more money, you can --- easier to do it and can …. But we have a way to --- to, you know, convince the community and continue to do it. So I am a collector. Actually have been persistent for more than 20 years. Now there are more and more people who believe and then, this going to become an important area. I am not as lucky as saying this double helix. The double helix --when this came out, that concept, right away people accepted it. There are some scientists like that because of source intuition. But there are some scientists, no, that’s impossible, but that impossible become possible. Including --- there’s another protein called ____. Another guy took a risk prior to that. At the very beginning, he was saying, say, the protein need to be ___ and then degraded. And when his concept came out, nobody believed it. They said, how come he is so stupid? He says that protein ____ 38:17. But that’s a regulation. You need to make a new one and then retire the old one. But at the very beginning, people didn’t believe it, said that’s stupid, why ___ but now it’s become central dogma.

T.A. Rosolowski, PhD:

Wow! Interesting.

Mien-Chie Hung, PhD:

Yeah. So this --- I mean, this cell surface receptor was in the cell surface, because of the time when it was discovered. But this cell surface can possibly send messages by relay, traditionally, but can also by itself. Translocate --- translo --- translocate to different location and do --- do a different function. So what’s wrong with that?

T.A. Rosolowski, PhD:

Yeah, yeah.

Mien-Chie Hung, PhD:

So what’s wrong with that? As long as it’s important. So --- so I …

T.A. Rosolowski, PhD:

Very interesting, yeah. And --- and very telling about the doing of science. You know …

Mien-Chie Hung, PhD:

Yeah. And then --- and I also feel that if it’s --- as long as reproducible, it’s real. One had to be persistent to carry out --- if you don’t want to --just because people don’t believe it, it’s not let’s don’t do it. Then what’s point to the science? If you just do anything that people believe just because they read the textbook. You don’t have to do anything. Now why don’t you create by yourself, so in the future you can contribute to textbook. And that --- when I say contribute to the textbook, I am by no means saying just contribute to textbook, because this is nature. And then, our next generation, our kids and human being need to know it, it’s nature. And they need to discover nature, that’s passing the knowledge, you know, to the future generation. We should not just do science because it’s easier to get funded, because it’s easier to publish. We do science because we want to discover nature. Yeah. So that’s all more --- more in fundamental science stuff.

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Chapter 13: A Twenty-Year Study that Promises a Paradigm Shift: The Yeast Two-Hybrid System

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