Cell Biologist Michael Overholtzer learned to ask questions about the natural world from an early age -- a skill that he still uses in his research into a process of cell-in-cell death known as entosis, which may prove to be a novel mechanism in tumor suppression.
I guess like most kids, I was very curious. I suppose this is one of the most appealing aspects of doing science, that one can entertain curiosity professionally. Looking back, I realize that much of my curiosity and creativity comes from my parents, who encouraged me to ask questions about everything. This was important, growing up, as I did, in a little town in eastern Pennsylvania called Gilbertsville. Being creative and imaginative opened up the world to imagine how things might work, biologically and otherwise. Clearly the way I was raised has a lot to do with my current profession, and sure enough my only sibling, my sister, became a scientist as well.
Music vs. Science
In 1992, I attended Ithaca College, a small, liberal arts school in upstate New York. At that time, my interests in science were matched by an interest in music. Much of the draw for Ithaca was provided by its fantastic music program. As it turns out, I am a much better scientist than musician -- although I did complete, just barely, a music minor in voice. Music remains a big part of my life.
As it also turns out, Ithaca has a fantastic biology program, and it was there that I met my first scientific mentor, Vicki Cameron. Vicki invited me to join her laboratory for summer research after my second year of college. I would later spend each summer of my undergraduate career and my entire senior year studying cellular respiration in yeast with Vicki. What fascinated me about this research was not the biological questions we were asking at the time but rather just being able to ask questions of biological systems at all -- and even occasionally getting answers.
It is ironic, I think, that I went to Ithaca for music, and, in retrospect, this decision probably determined that I would stay in science. Because I was at a small school, I had the chance to get into the lab and play an important role in research. As there is no graduate program, undergraduates really drive the experimentation along with their mentors. It also provides the chance to develop a very close mentoring relationship, which I had with Vicki, who was a wonderful teacher and role model. She was the first of three strong personalities that really shaped me into the scientist that I am today. At Ithaca, having the freedom to do my own experiments, to follow, scientifically, wherever my curiosity led me, was wonderful. I was addicted.
Even with my new-found addiction, by the time I graduated from Ithaca, I was not absolutely certain I wanted to go to graduate school. I'm not someone who spends a lot of time planning ahead and thinking about my life. I didn't necessarily have the career goal in mind of being a professor at a large institution. For me, science is more about passion. I think I've been a scientist my whole life, and deciding to go onto graduate school really just allowed me to continue doing experiments.
Big Thinking
In 1996, I decided to go to Princeton University to get my PhD because I was really excited about the science being done there. I ended up rotating into the lab of Arnold Levine, who started me thinking about cancer, and who ultimately became the most important mentor in shaping my scientific personality. He is such a big thinker and a leader in cancer biology, but also a wonderful person. It was easy to get swept up into the kinds of questions he was asking in his laboratory.
Halfway through my graduate career, Arnold moved to Rockefeller University and I went along with him. Having grown up in a small town and spending both my undergraduate and graduate careers in smaller communities, the move to New York was quite an experience. Within two weeks, I was in love with the city. Literally, within less than a month, it had become my favorite place.
At Rockefeller, I tried a number of different research projects, before settling on investigating the role of p53 -- a protein that regulates the cell cycle and cell death, as well as acting as a tumor suppressor -- in genome stability. More specifically, we started to look at human tumors to better understand the role of p53.
There were aspects of that research that became very important to my career, including developing collaborations with investigators here at Memorial Sloan-Kettering Cancer Center, which was right across the street. The real reasons why you perform certain experiments in the lab become much clearer when suddenly you are in an environment where there are actual cancer patients in close physical proximity to you. It became very motivating for me. I realized that this career is not just about satisfying my own curiosities, this career is about trying to help people.
We looked at genomic instability by trying to map new oncogenes, or tumor suppressors, which might be amplified or deleted in a genome. It became a little frustrating to learn just how "noisy" genomic instability can actually be. Plus, there was a shortage of good in vitro assays to use to understand whether any of these candidate genes could even have any relevant functions. If you have an amplified region of the genome, you can have hundreds of genes in there to sift through. This started me thinking about a wholly different kind of postdoctoral research.
3-D Cultures
Then in 2002, I chose to work in Joan Brugge's lab at the Harvard Medical School. Joan, now Chair of the Department of Cell Biology at Harvard, pioneered a model in which mammary epithelial cells are placed in a three-dimensional culture and used to mimic breast tumors. The idea is that instead of growing these cells on a flat, plastic surface, these cells are grown in an architectural structure closer to what is found in the body. To me, this was attractive because it allowed you to look at a number of different kinds of phenotypes that more closely mimic what breast cancer tumors actually do in the body when compared with what could be done with standard tissue culture techniques. Also, like Arnold Levine, Joan is a wonderfully imaginative thinker and a leader in cancer biology. This felt like the right next step for me.
I began my work with Joan where I had left off with Arnie, looking for genes that might be important for cancer by their amplification or deletion in the genome, but now applying the three-dimensional-type culture model to phenotypically screen these candidates to see how they would make cells behave. It was a bit like a fishing expedition, in that it was a long and at times frustrating ordeal. But it eventually lead to my first paper with Joan, for a project that I'm still working on involving what is known as the Yes-Associated Protein, YAP.
YAP can bind a number of transcription factors, including those of the p53 family, and, in the process, regulate their activation. YAP is becoming increasingly important as it is clear that this protein, and the Hippo pathway that regulates it, has a role in cell proliferation and survival in mammalian cells, which means that they are likely deregulated in a host of cancers. It is interesting, I think, to point out that we weren't looking to find YAP, per se. We were fishing and YAP bit on the line. You never know what you might find in research, but if you are asking the right kinds of questions, often the most unexpected turns are the true gifts that come from the long, and often frustrating process.
A Peculiar Observation
The research that I am focusing on currently derives from an unusual observation of cell behavior that we noticed, by chance, while working on another project. We noticed what appeared to be one breast cell completely encapsulated inside of another. And then we kept noticing this occurrence. It was very peculiar. We continued to see it, and I created a small side project to try figure out what it was. When we had enough data to show to a small group of collaborators, one of them, a pathologist remarked that this had been seen before, in human cancers. They even had a name for it, cannibalism, or cell-in-cell. When we saw images of cell-in-cell from human cancers, the images looked exactly the same as ours. To see it in the lab, and then to discover that it is actually happening in the clinical setting was extremely exciting.
The way in which we learned to induce these cells in the lab to do this process also turned out to be very much like what is going on in patients. We found that these cell-in-cell structures can form by a cell adhesion-based mechanism, which we named entosis. And we also found that one could induce this process by detaching cells from the normal protein matrix beds on which they like to sit -- a situation not unlike what is going on in some human cancers, where tumor cells grow away from their normal environments and must adapt to life on altered or absent matrices. In fact, these structures can be a feature of tumor cells found in fluid in the body, and all along we were forcing cells into a fluid-like state to induce entosis in the lab.
Cell-in-Cell Structures and Cell Death
Although cells internalized by entosis are initially alive -- they can actually divide within or be released from the host cell -- the majority of internalized cells eventually undergo cell death. Cell death occurs by an unusual mechanism involving total cellular degradation inside of a large lysosomal compartment. It is conceivable that this mechanism of cell-in-cell formation could suppress primary tumor growth, or could inhibit metastatic lesions from forming outside of their natural environment.
From a scientific career perspective, my attitude has always been to immerse myself in the science and let the long-term plans for success take care of themselves. Which I take as meaning, don't get too caught up in what you hope will happen. However, throughout all this, I began to realize the importance, for me, of my laboratory research being carefully paralleled with patients.
To do this, I would need access to human tumor samples, and I wanted to continue to pair my thinking in the lab and what I see in my microscopes with what pathologists see in human cancers in the microscope everyday. As a result, I knew that I would need to work in the context of a cancer center. And what more perfect place than Memorial Sloan-Kettering? Alan Hall, who chairs the Cell Biology Program at Sloan-Kettering Institute, has a remarkable reputation and when the position in the program became available, I realized that it was my dream job. When I was offered an opportunity to join, I canceled all of my other second interviews at other places because this is where I wanted to be.
A Unique Opportunity , A Great Responsibility
Now, in the short term, the challenge is to see where this entosis project will lead. I would like to understand its clinical relevance for cancer. It is both a unique opportunity and a great responsibility. To do this I have three specific research goals. First, I hope to further characterize the mechanism of entosis by examining in much more detail how these unusual cell-in-cell structures form. Second, I would like to examine the cell death mechanism of internalized cells. This includes understanding what triggers an internalized cell to die, the mechanisms that execute the cell death process, and the contributions of inner vs. outer cells to cell death. And, third, and most important, I plan to examine the role of cell-in-cell formation in tumorigenesis, which will involve studies to inhibit or promote entosis in order to examine its effect on tumor growth and progression, both in vitro and in vivo.
I do feel a responsibility to the patients with cancer across the street in Memorial Hospital. Some basic researchers may say that if you're in this to cure cancer, you haven't been doing this sort of research for very long -- meaning that if you are in this to cure cancer, you are naïve. But I tend to disagree. Because we have noticed something that does take place in cancer patients' tumors, it gives us hope that what we are learning here in the lab could affect patients' lives somewhere down the line. For me, that is why I do this. I want my lab to be driven with great curiosity and imagination, and to study unusual or new ideas, but always with the larger goal of sifting through these new ideas to understand whether what we are studying could be important for human disease.
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