Cell Adhesion, Signaling and Cancer

Part 1: Adhesion, Signaling and Cancer

00:00:03.10 Hello. My name is Mary Beckerle, and I'm a professor of biology and oncological sciences
00:00:07.27 at Hunstman Cancer Institute and the University of Utah.
00:00:10.23 It's really a pleasure to be with you today,
00:00:13.15 wherever you are, to talk with you about adhesion and signaling,
00:00:18.26 critical cell behaviors that control a variety of important cellular
00:00:26.00 processes, and in particular focus on the role of adhesion and signaling in cancer biology.
00:00:32.11 My presentation is going to be divided into three parts
00:00:36.14 and the first part I am going to provide a very brief introduction to
00:00:40.15 the cancer problem and our appreciation of the mechanisms
00:00:45.19 underlying the development of cancer, with particular emphasis
00:00:48.28 on the role of cell adhesion in tumor development.
00:00:51.26 In the second part I am going to tell you a short story from work in my own laboratory
00:00:57.12 about the discovery of a molecule that is found at sites of adhesion
00:01:00.29 and how we now appreciate that this protein may regulate
00:01:06.03 processes that are important
00:01:07.17 in a particular type of cancer, Ewing's sarcoma.
00:01:10.20 And finally I am going to tell you about
00:01:13.28 some very exciting new results, which implicate
00:01:17.19 specialized zones where cells attach to the extracellular matrix
00:01:22.05 and the ability of cells to sense physical forces.
00:01:25.25 And these physical forces, again, influence cell behavior in really important
00:01:32.23 ways, and we are just at a sort of new frontier in terms of
00:01:36.09 understanding how cells perceive and respond to mechanical cues.
00:01:40.27 So in part one of my talk, I am going to again give you
00:01:45.21 a brief introduction to the cancer problem.
00:01:48.23 I think everybody appreciates what a large problem
00:01:53.00 cancer is from a human health perspective.
00:01:55.04 All of us have been touched by cancer in one way or another,
00:01:57.14 either with someone we know, a family member or a friend,
00:02:01.17 encountering this illness. It is a major
00:02:06.03 global health problem with over 6 million deaths worldwide
00:02:11.29 from cancer on an annual basis.
00:02:14.25 In the United States alone we lose one person to cancer every minute of every day.
00:02:21.07 And there are over 3,800 people diagnosed with cancer each day.
00:02:26.03 You can see from these data that more than 20 million new
00:02:30.11 cancer diagnoses have occurred since 1990, which means
00:02:33.25 that many, many more people are living with this
00:02:36.23 disease. Worldwide it is anticipated that half of all men
00:02:41.26 and a third of all women will get cancer at some time in their lifetimes.
00:02:46.20 So this is clearly a major medical challenge worldwide,
00:02:51.19 and really research is the hope for the future
00:02:54.21 to impact cancer detection, diagnosis, treatment and ultimately prevention.
00:03:01.20 We already know that some cancers can be prevented.
00:03:06.03 Smoking, for example, accounts for at least 30% of all cancers,
00:03:09.22 and in the case of lung cancer, almost 90% of lung cancer deaths
00:03:14.00 can be attributed to smoking.
00:03:16.08 Similarly, excessive sun exposure, including the use of tanning booths,
00:03:21.23 and excessive sun exposure in the absence of sun protection
00:03:26.26 is clearly responsible for many types of skin cancer, including
00:03:30.23 a very lethal form of skin cancer called melanoma.
00:03:34.10 So just by changing our behavior, minimizing or eliminating smoking
00:03:40.27 and minimizing or eliminating exposure to sunlight in the absence of protection,
00:03:48.09 can have a huge impact on the occurrence of these devastating cancers.
00:03:52.27 Similarly we now have a behavioral intervention, a new vaccine against
00:03:57.27 human papilloma virus, which we believe will have a huge impact
00:04:03.18 in reducing mortality due to cervical cancer.
00:04:07.14 So science has already taught us a lot about how to prevent cancers.
00:04:11.23 And it's also taught us that early detection of cancer
00:04:16.06 is really critical. We know that cancer acquires
00:04:20.16 more and more aggressive potential as time goes on.
00:04:24.04 and those more aggressive tumors are much more difficult
00:04:28.27 to treat, often widely disseminated throughout the body.
00:04:31.29 So screening strategies that can detect cancer early
00:04:36.04 before it has begun to spread can have a huge impact on the ultimate outcome.
00:04:40.29 So in many cases, in the case of breast, colon cancer, prostate cancer,
00:04:46.27 melanoma, skin cancer, and others,
00:04:49.12 screening processes are readily available,
00:04:51.18 and a huge human health burden could be readily eliminated
00:04:54.15 by individuals really adhering to screening guidelines
00:05:00.00 and taking advantage of these opportunities
00:05:02.11 to detect cancers very early before they become more lethal.
00:05:07.27 So really what is the base of cancer? How does cancer develop in the first place?
00:05:14.19 Well, we really have appreciated over that past 20 or 30 years that cancer is a cellular
00:05:20.26 disease that results from derangements in control pathways that regulate cell number.
00:05:28.26 Here you can see a nice illustration of how the human body
00:05:33.00 is comprised of functional units called cells,
00:05:37.09 trillions of cells in the body. And these cells are really organized in beautiful
00:05:43.11 patterns when they are grown in culture, and as you'll see,
00:05:46.29 are also very well organized in situ.
00:05:50.19 Here's an example of a section through
00:05:54.12 the stomach where you can really appreciate the highly ordered
00:06:00.06 structure of the tissue,
00:06:02.05 with the individual secretory cells relating to each other
00:06:05.16 with apical and basal aspects, with a nicely organized lumen,
00:06:11.25 with each of these secretory structures looking very similar to each other,
00:06:17.17 really illustrating that cells have information
00:06:21.11 that establish pattern and morphology, and that they are receiving signals
00:06:27.02 and controlling their architecture under normal conditions.
00:06:30.07 Things are very different in the case of tumors.
00:06:34.12 And here you can see a lovely illustration showing
00:06:37.15 the difference between normal mammary gland and a mammary tumor.
00:06:42.06 In the top panel you can see mammary gland with a milk duct
00:06:46.28 and, again, these cells are nicely organized with a lumen here and surrounding
00:06:52.09 stromal tissues, and look what happens in breast carcinoma.
00:06:57.16 We've completely lost the architecture of the tissue.
00:07:01.05 The cell number, if we were to analyze it, would be out of control,
00:07:04.27 but fundamentally the cells have lost their capacity
00:07:08.28 to recognize signals from their surroundings
00:07:12.18 and to acquire this highly ordered, differentiated phenotype.
00:07:18.20 The fundamental thing, the first thing that happens
00:07:23.04 in the establishment of tumors is of course derangement in the control of cell number.
00:07:29.20 And cells begin, as you can see in this diagram,
00:07:33.15 to grow out of control, and that then disturbs the architecture.
00:07:37.23 And this growth, or loss of growth control
00:07:42.16 can happen at a number of different levels illustrated schematically here.
00:07:47.20 And I'll just touch on a couple of them today.
00:07:50.09 You can...tumor cells can acquire an insensitivity to antigrowth signals,
00:07:58.15 to signals that normally constrain their
00:08:01.10 proliferation, and an example of that is the inability to recognize
00:08:06.14 that they're surrounded by other cells.
00:08:08.22 Their inability to really detect or respond
00:08:12.19 appropriately to environmental cues
00:08:15.04 that normally would slow their growth.
00:08:20.16 A second example is that they can become self-sufficient and not require positive growth
00:08:26.24 signals anymore. Normally cells divided only when there are growth factors
00:08:31.11 present that activate signaling pathways
00:08:33.23 that lead to proliferation and cell division.
00:08:36.05 In tumors, many times the cells have lost that need
00:08:42.00 to have exogenous growth signals,
00:08:45.14 and they're sort of constitutively activated for the proliferation pathways.
00:08:49.24 And finally some recent work just in the last 10 to 20 years
00:08:56.03 has revealed that all of the changes in cell number that occur
00:08:59.26 in cancers cannot be attributed just to regulation of proliferation,
00:09:03.22 but rather another very important mechanism
00:09:06.25 by which cell number is controlled is through a process called apoptosis
00:09:11.05 or programmed cell death.
00:09:13.20 And programmed cell death is a process that is used
00:09:17.18 during development, it is used during normal tissue and organ maintenance, and
00:09:23.10 if that process becomes curtailed in some way, cells that should normally die
00:09:29.26 will fail to die, and of course that can also give rise to enhanced cell number.
00:09:34.13 And this is also something that can cause cancer.
00:09:41.06 Fundamentally all of these behaviors of cells,
00:09:43.25 the ability of cells to get signals from their environment
00:09:47.18 the ability of cells to interact and to acquire this highly ordered architecture
00:09:53.24 within tissues is completely dependent on our genetic material,
00:09:58.27 which really controls cell behavior.
00:10:01.25 And here you can see the diagram of a cell
00:10:06.28 or an image of a cell where
00:10:08.08 the red illustrates the cytoskeleton, and here is the nucleus.
00:10:12.24 And the nucleus of course contains all of the genetic material: 23 pairs of chromosomes,
00:10:19.02 which represent or are comprised of the genetic material,
00:10:24.08 DNA, organized into units called genes.
00:10:29.06 And the human genome project has led to an appreciation
00:10:33.16 that humans have about 30,000 protein coding genes,
00:10:37.29 and you can see here that that effort has allowed us
00:10:42.03 to appreciate not only what those genes are
00:10:45.14 and what they encode, but also where they are located on each chromosome.
00:10:49.10 So here are the sex chromosomes, the X and Y chromosomes.
00:10:52.04 And here's a pattern that illustrates the distribution of the genes
00:10:58.12 on those two chromosomes. We now appreciate that it is alteration in genes, the
00:11:05.24 genetic material, that really is responsible for the early changes that lead to cancer.
00:11:10.19 Normal genetic material in a healthy individual leads to control of cell growth and cell death
00:11:20.08 and all of the behaviors of cells that I have described previously.
00:11:25.01 And we now understand that it is lesions in the genetic material,
00:11:29.25 the DNA, and mutations in those genes that alter those important regulatory pathways
00:11:36.16 and lead to excessive cell growth and ultimately to a tumor.
00:11:42.11 Excessive cell growth, or inadequate cell death and then the tumor.
00:11:46.07 These genetic changes can be inherited and passed from
00:11:50.25 parent to child or they can be acquired over time by exposure to chemical carcinogens
00:11:58.12 too much sunlight, etc. We now have a much deeper understanding
00:12:05.12 of some of the basic causes, the basic genetic causes, that give
00:12:10.20 rise to tumors and this understanding has come from a variety of approaches.
00:12:15.11 It has come from trying to recreate a cancer phenotype, looking at cells in culture,
00:12:22.06 and it has also come from direct analysis
00:12:25.28 of patients and their families where there is an inherited sensitivity
00:12:32.00 to the development of cancer.
00:12:33.26 And I'll just give you a couple of examples of this approach
00:12:37.03 and the powerful impact that this new genetic understanding
00:12:41.07 has on our ability to think about creative ways to address this clinical problem.
00:12:46.19 We know as I mentioned that some cancers run in families,
00:12:51.24 you've heard about friends or colleagues perhaps who have a high probability of colon cancer
00:12:58.21 in their family. And although this doesn't happen very frequently we now can
00:13:04.28 actually look at populations of individuals and identify these inherited patterns.
00:13:13.17 These high predisposition to particular types of cancer and one really unique resource
00:13:19.14 that allows us to do this is the Utah population database,
00:13:23.15 which has extensive pedigree information going back many generations
00:13:28.09 and over 20 million records linked to the pedigree information, so
00:13:33.01 we have a very, very clear picture of clinical information linked to the family structures.
00:13:44.16 And with that approach one can identify families for example that have a high probability of
00:13:52.25 developing colon cancer, and then actually search through the DNA provided by individuals
00:14:00.11 in those families for the particular segment of DNA that tracks with the inherited
00:14:07.22 predisposition to colon cancer. And in that type of strategy
00:14:11.24 many human disease genes have been identified,
00:14:15.08 and these include the colon cancer gene, APC, as well as breast cancer genes,
00:14:21.02 BRCA1 and BRCA2, and the melanoma gene, p16.
00:14:25.15 It's actually extremely useful to have this genetic information
00:14:32.12 in terms of developing better strategies for detection, treatment, and ultimately
00:14:38.22 prevention of cancer. Let me just give you an example
00:14:41.05 from colon cancer, which I already mentioned was identified
00:14:46.01 in patients that have a predisposition to this familial form of cancer
00:14:52.15 using this Utah population database.
00:14:54.24 And it was also identified independently in sporadic tumors by other investigators as well.
00:15:02.04 But here you can see the highly organized structure, diagrammatically
00:15:07.15 of normal colon. And really the initiating step in colon cancer is
00:15:12.27 mutation of the APC gene. We know now that this is the cause of these inherited forms
00:15:18.27 of colon cancer, as well as is responsible for the initiation in the vast majority
00:15:23.24 of sporadic colon cancers as well.
00:15:26.28 It's, as I mentioned, over time these initiated cells
00:15:35.07 can develop additional changes, which can lead
00:15:38.25 to the beginnings of uncontrolled growth, and at that stage
00:15:42.07 in a human patient you would have what's called an adenomatous polyp,
00:15:47.08 a small growth of cell material associated with the wall of the
00:15:52.14 colon. But this tumor, this is not yet
00:15:55.10 a cancer, it is an adenoma. It is not invasive, and it is easily removed
00:16:00.00 surgically. Over time, however, you can see that the tumor can progress
00:16:04.12 acquire properties where it can invade and become a carcinoma
00:16:09.10 and with additional mutations even begin to spread or metastasize.
00:16:13.24 And of course at these stages these tumors are much more difficult to
00:16:17.17 treat. They are widely disseminated. They have invaded the wall of the bowel,
00:16:22.09 and this is then very, very challenging from a clinical perspective.
00:16:26.12 So knowing that you, for example, are in a family where colon cancer
00:16:32.05 is prominent and potentially inherited, you can go and have genetic testing, which
00:16:39.23 if you are then a carrier of the disease susceptibility gene,
00:16:43.27 the mutant form of APC, you will be guided
00:16:47.25 to much more robust screening strategies,
00:16:51.16 and that will allow the detection of the developing cancers
00:16:55.15 at this adenomatous polyp stage early enough that it
00:16:58.17 can be removed for a complete cure.
00:17:02.15 So knowing, and being able to screen and identify individuals
00:17:07.06 who are at high risk for colon cancer clearly
00:17:09.24 has an impact on their health, and in addition,
00:17:16.02 knowing that APC is the first...that mutations in APC represent
00:17:20.23 the first step in the development of this disease
00:17:23.00 we can then go back into the laboratory and really try and dissect
00:17:26.25 the pathways that are controlled by APC and use that information potentially to develop
00:17:34.18 therapeutic interventions.
00:17:37.12 And we know now a lot about the function of the APC protein,
00:17:41.05 and one really exciting new development has come
00:17:45.07 from the work of David Jones, which has clearly illustrated
00:17:49.27 that the APC is required for retinoic acid metabolism.
00:17:53.11 And he has been able in some preclinical studies to provide retinoic acid
00:17:58.13 derivatives to whole organisms and reverse the course
00:18:03.08 that would have occurred due to the APC mutation.
00:18:07.18 And so again, knowledge of the fundamental genetic change that occurs in a tumor
00:18:14.08 the initiating change, can allow us to, 1: identify individuals who are at high risk
00:18:19.23 for developing a tumor, and 2: with cellular and molecular analysis
00:18:25.07 of the role of that normal protein product
00:18:30.29 help us to identify strategies that will lead to changes in the outcome for patients.
00:18:40.19 Now another thing that has, I think, really been striking
00:18:43.24 from the genetic analysis of tumors both
00:18:46.19 in a classical genetic sense and also in a molecular genetic sense
00:18:50.07 is that different genes are turned on in different tumors.
00:18:54.18 I think in 1971 when Richard Nixon announced the war on cancer
00:18:58.16 scientists, as well as the public,
00:19:01.04 were very optimistic that we were going to identify a single gene or a handful
00:19:06.08 of genes, or a single cause or a handful of causes
00:19:09.12 for cancer, and that we would then be able to identify some sort of silver bullet
00:19:14.08 that would allow us to eliminate this disease. But we now really appreciate that
00:19:20.07 cancer is hundreds of diseases. It is a much more complex challenge.
00:19:24.23 I have already given you an example of several cancer mutations
00:19:28.16 that can cause different kinds of cancers,
00:19:30.14 and we now have the capacity to actually look at all 30,000 human genes
00:19:36.15 for their expression simultaneously
00:19:38.08 using microarray strategies, and when we do this... in this image here
00:19:44.12 we are looking at 1,800 genes running from the top to the bottom
00:19:48.01 of this panel and 140 or so different types of tumors running from left to right.
00:19:54.24 And what you can see when you analyze this
00:19:58.02 with the green genes being... green spots
00:20:00.16 representing genes that are turned off
00:20:03.09 and the red spots representing genes that are turned on,
00:20:06.06 is that these patterns of gene expression actually segregate into groups.
00:20:12.12 And many times you can see that, for example here,
00:20:14.26 all of these prostate cancer samples have very, very similar patterns
00:20:20.24 of expression and that...but on the other hand,
00:20:25.08 if you look down here in breast cancers you can see that
00:20:28.21 even though these in the box here are all breast cancers, genes expressed in breast cancers,
00:20:34.16 you can see that they are not all green and they are not all red,
00:20:37.10 illustrating that there is a great deal of heterogeneity even within a particular type of tumor.
00:20:43.10 And this is really one of the major challenges
00:20:46.27 I think for cancer biologists and clinical care providers
00:20:50.28 that we are facing currently. It is very
00:20:53.17 common for two women with breast cancer to have the same diagnosis,
00:20:57.16 the same treatment, and
00:20:59.04 have very, very different outcomes.
00:21:00.29 One person may succumb to their cancer
00:21:02.21 within a few months, another may live for ten years,
00:21:06.02 and we really now can appreciate
00:21:09.08 because of this kind of detailed molecular understanding
00:21:12.02 molecular fingerprinting analysis, that we really need
00:21:16.04 to have a more high resolution view of the tumor itself,
00:21:20.27 in order to define the most appropriate treatment for that tumor.
00:21:25.26 So I think a really major revolution in our thinking
00:21:30.28 about cancer biology is this appreciation of
00:21:35.03 the diversity both of tumor populations and of patient populations and this has
00:21:41.28 really come specifically through advances in genetics,
00:21:46.04 molecular genetics, and the approaches that I have described today.
00:21:50.05 We used to think that all tumors were the same
00:21:53.00 and a tumor in any individual context was the same, we now
00:21:57.16 appreciate that there are many, many
00:21:59.11 differences in tumors, even tumors of the same tissue type,
00:22:04.20 can be extremely different in terms of their properties,
00:22:09.00 and therefore extremely different in terms of their responses to therapeutic intervention.
00:22:12.27 And we also appreciate that the tumors within the context of an individual
00:22:19.00 the same, even the same type of tumor within the context of a
00:22:21.29 different individual can be very different as well.
00:22:24.25 And we've had a presentation in this series from
00:22:29.23 Brian Druker which addresses that exact issue.
00:22:33.20 So, I think that again this revolution in molecular genetics that we've seen
00:22:43.14 occurring just very recently really gives us
00:22:46.28 increased genetic understanding of the causation of cancer,
00:22:50.07 makes us appreciate how diverse the mechanisms are that can cause cancer,
00:22:56.21 but gives us a lot of hope that with that information
00:23:00.12 we can be empowered to use new strategies to address this really major health issue.
00:23:07.11 We can, as I've described, begin to
00:23:11.03 identify individuals who are at high risk for developing certain types of cancers
00:23:16.00 particularly in the inherited forms of cancers,
00:23:18.24 but maybe over time the sporadic ones as well, and then with that predictive power
00:23:25.24 develop guidelines for more aggressive screening and early detection.
00:23:30.26 And then the goal would be of course to then prevent those tumors from ever
00:23:34.20 developing or at least screen and catch them early before
00:23:38.15 they are very dangerous.
00:23:39.09 And really this all leads to this view of a more personalized approach
00:23:44.00 both in terms of identifying individuals at risk, as well as,
00:23:48.24 using molecular strategies for high resolution diagnosis of tumors, so that
00:23:54.22 the tumors can be matched in a very rigorous and appropriate way
00:23:59.09 to therapeutic intervention.
00:24:02.12 and moreover, that the individual themselves can be identified
00:24:10.06 for individuals that may be at risk for adverse events
00:24:15.09 from various chemotherapeutic interventions, etc.
00:24:18.25 So I think we are moving into a new era
00:24:22.04 in cancer biology that has great promise for the future.
00:24:27.12 We do have to face the fact that this is a very complex situation, and that
00:24:34.15 the processes of control of cell growth and cell death
00:24:38.17 are regulated by many, many biochemical pathways.
00:24:42.05 This diagram illustrates just some of the pathways
00:24:45.29 that are involved in controlling the circuits that regulate cell growth
00:24:51.17 and cell death and can contribute to cancer.
00:24:53.22 All of the proteins marked in red here,
00:24:56.17 here is APC, here, over here is p16 that I told you was responsible for melanoma,
00:25:03.06 and you can see that all of the elements that are marked in red
00:25:07.19 have already been identified as having a role in cancer development.
00:25:11.19 And so there are many, many ways that cancer can develop,
00:25:13.23 and therefore there are going to have to be many, many ways
00:25:15.28 to try to tackle the individual cases of cancer.
00:25:21.15 The first human oncogene that was identified is the Ras protein,
00:25:28.27 and this was identified in a screen for
00:25:33.08 cDNAs that could cause cellular transformation in a cell-based assay.
00:25:38.17 And it was very, very striking at the time that
00:25:42.25 this gene...a single amino acid change in the Ras...
00:25:47.17 that gave rise to a single amino acid change in the Ras protein
00:25:51.00 could be responsible for the establishment of bladder carcinoma.
00:25:55.02 And we now have a very deep appreciation of exactly what pathways are
00:26:01.17 regulated by Ras. So here you can see the Ras protein is part of a critical signaling pathway
00:26:11.17 that starts with growth factor exposure and ends with regulation of cell cycle control.
00:26:20.22 And we now understand that manipulation of this pathway,
00:26:26.06 either by direct mutation of the gene that encodes Ras,
00:26:29.13 or also of any of the components in this pathway
00:26:33.24 can lead to tumors.
00:26:37.08 So for example in glioblastomas, we have an increase in the growth factor PDGF,
00:26:48.03 which seems to play a really important role in the development of that type of tumor.
00:26:52.19 Similarly, an increase in TGF alpha expression is prominent in certain sarcomas.
00:26:59.25 So you can see that you can control the activity of
00:27:03.12 this pathway at the level of production of growth factors.
00:27:07.08 In addition tumors may have altered growth factor receptor profiles or activities
00:27:13.18 that are responsible for the development of a particular tumor type.
00:27:20.01 One really common example is the EGF receptor,
00:27:23.13 which can either be upregulated by activating mutations
00:27:27.26 or upregulated by increased copy number,
00:27:31.12 and this is another way to, of course, activate this critical pathway which controls
00:27:38.00 cell growth and confer to cells self-sufficiency with respect to growth
00:27:42.08 signals. We've already discussed the activation of Ras itself.
00:27:47.20 Activating mutations in Ras can turn this
00:27:50.22 pathway on, out of control, and lead to uncontrolled cell proliferation.
00:27:56.18 And interestingly there are also other co-factors that can
00:28:01.18 be altered to give rise to uncontrolled growth
00:28:06.11 and uncontrolled activation of this pathway.
00:28:08.07 And one example of this is the integrins which are receptors for the extracellular matrix.
00:28:14.29 These integrins are transmembrane heterodimeric cell surface receptors,
00:28:20.14 and they were identified initially in an antibody
00:28:23.19 screen in which investigators were looking for
00:28:28.07 monoclonal antibodies that when applied to cells would
00:28:31.20 cause them to lose their ability to attach to extracellular matrix.
00:28:35.23 And those antibodies ultimately allowed the identification
00:28:40.03 of this very important family of cell surface proteins.
00:28:46.08 These integrins are critical for adhesion to extracellular matrix,
00:28:51.02 as well as transmembrane signaling, and they are concentrated
00:28:54.12 in cells at specific zones called focal adhesions,
00:28:58.17 which are specialized areas of the plasma membrane
00:29:01.23 that are important for adhesion to extracellular matrix.
00:29:08.27 You can see an example of one of these focal adhesions by electron microscopy
00:29:15.05 in this image and what we appreciate is that these areas
00:29:20.13 actually of enriched, enriched in integrins
00:29:24.09 where the cells attach to the extracellular matrix
00:29:26.11 are areas where the extracellular environment
00:29:29.08 is really aligned and integrated with the
00:29:33.00 intracellular environment, in particular the cytoskeleton.
00:29:36.02 So these receptors actually form a link between the extracellular matrix and
00:29:41.00 the cytoskeleton, and there are a variety of proteins that are present
00:29:46.04 clustered with the integrins at these sites which facilitate the linkage
00:29:50.04 of the integrins on the membrane to the actin cytoskeleton.
00:29:53.22 And although I don't have time to discuss this now,
00:29:55.29 this linkage is actually quite critical for the function
00:29:59.15 of the integrins both in terms of adhesion as well as in terms of signaling.
00:30:05.10 So we now know from studies of integrins
00:30:09.05 that these are signaling receptors not passive
00:30:12.13 adhesion receptors, but really active
00:30:15.29 classical receptors that respond to extracellular cues
00:30:19.18 and transmit information bi-directionally across the
00:30:22.28 plasma membrane to regulate both the ability of the cells to
00:30:26.24 adhere as well as their ability to migrate, which is, of course, critical for metastasis
00:30:33.27 in tumors, as well as to proliferate and to survive.
00:30:38.15 So why would these adhesion receptors have an impact on cell growth?
00:30:45.25 We have talked about the fact that growth factors are a critical signal
00:30:50.04 that regulates cell proliferation, particularly via the Ras signaling pathway.
00:30:54.22 And why would these cell adhesion receptors have an impact on cell growth?
00:31:00.17 Well, it turns out that integrin dependent adhesion
00:31:04.03 is actually required for a normal growth factor response.
00:31:07.25 And this became evident in a really lovely experiment
00:31:10.28 from Martin Schwartz's lab in the late 1990s where he
00:31:15.02 looked at the response of cells to exposure to growth factor.
00:31:20.18 And he could follow this by looking at the activation of one of the MAP kinases
00:31:27.07 in the signaling pathway, and here you can see
00:31:31.09 lots of activated ERK signaling, and over here, not very much.
00:31:38.29 And what he did was to take cells either in suspension or
00:31:43.14 attached and expose them to growth factor, and what you can see down here is that
00:31:48.10 the cells exposed to growth factor that were attached
00:31:53.07 launched a really robust signaling response,
00:31:57.08 activation of this MAP kinase signaling pathway,
00:32:00.10 whereas the same cells devoid of integrin engagement
00:32:05.22 and held in suspension, when they were exposed to growth factor
00:32:10.23 a very, very minimal signaling response
00:32:13.25 occurred. So this was really one of the first indications that
00:32:17.10 it's not as simple as a growth factor receptor on the surface of a cell,
00:32:22.00 that in fact there is a lot of cross talk and cells are monitoring also
00:32:25.23 whether they have integrins engaged and whether they are attached
00:32:28.29 in order to divide. And we now know that the integrins
00:32:32.28 actually talk to this canonical growth factor signaling pathway
00:32:37.02 at a number of different levels to regulate
00:32:41.02 its activity. They can be directly linked to the growth factor receptor.
00:32:46.20 They can regulate some of the MAP kinases,
00:32:50.13 and they can even act at the end of this pathway to control
00:32:53.26 whether one of the final activating proteins in the pathway can get
00:33:00.11 into the nucleus efficiently to act to regulate cell proliferation.
00:33:05.02 So again, this was a very exciting observation that integrins
00:33:10.01 are really critical for growth factor signaling and play
00:33:13.23 a role in controlling cell growth,
00:33:16.12 and then of course, led to the idea that perhaps if we could control
00:33:20.29 the integrins and their signaling behavior, we might be able to control
00:33:26.12 cell growth. And since these molecules are on the cell surface
00:33:29.08 they are easily accessible to agents that
00:33:32.22 can be provided on the outside of cells,
00:33:36.14 which makes them a good potential therapeutic target.
00:33:40.12 And so here is a preclinical study in which anti-integrin antibodies
00:33:45.28 were used to explore the impact on proliferation of ovarian tumor cells.
00:33:51.26 And here you can see in the open bar,
00:33:55.16 we are looking at ovarian tumor cells with a control antibody,
00:33:59.29 and that has no effect on growth, and the cells
00:34:02.04 continue to grow even at high antibody concentrations.
00:34:05.09 However, with the filled bar you can see
00:34:09.09 that as you increase the amount of anti-integrin specific antibody, the proliferation
00:34:16.18 of these ovarian tumor cells in culture diminishes
00:34:20.14 really dramatically. So this has given a lot of hope to the idea that
00:34:24.19 one might be able to control tumor cell growth
00:34:29.04 by manipulating integrin either directly,
00:34:33.29 or manipulating some of its downstream effectors,
00:34:37.26 which we now know are quite complex.
00:34:40.28 But I think that there's a lot of promise here, and we already have now
00:34:45.07 several integrin inhibitors in clinical trials, both antibodies as well as small peptides
00:34:52.23 for a variety of cancer applications.
00:34:55.27 So I am going to just briefly summarize then this broad introduction,
00:35:02.09 which I hope I have convinced you that cancer is a
00:35:05.16 genetic disease. That it is a quite complex disease where many genes
00:35:10.07 can be mutated to give rise to cancer because there are
00:35:12.29 so many pathways, so many critical cellular biochemical pathways that
00:35:17.20 control cell growth and cell death.
00:35:20.27 And among those, somewhat surprisingly, are the pathways
00:35:26.15 that are involved in cell adhesion. And really
00:35:30.06 I think that we are now at an inflection point in our
00:35:34.02 ability to utilize the wealth of information we have
00:35:38.22 about human genetics, cellular pathways, to have an impact
00:35:42.22 on the establishment of new therapies
00:35:45.23 that can ultimately impact human health by
00:35:48.25 affecting cancer development or establishment
00:35:55.13 or progression. Thank you.

Part 2: Discovery and Characterization of a Focal Adhesion Protein Implicated in Tumor Progression

00:00:03.26 Hello, my name is Mary Beckerle,
00:00:05.24 and I'm a professor of Biology and Oncological Sciences at
00:00:08.26 Huntsman Cancer Institute at the University of Utah.
00:00:11.04 In this part of my presentation, I want
00:00:15.13 to describe to you some work in my laboratory
00:00:19.07 on the discovery and characterization of a focal adhesion
00:00:22.22 protein that is implicated in tumor progression.
00:00:28.01 As we've discussed in part one, cells are surrounded by
00:00:34.19 other cells and extracellular matrix
00:00:37.05 material and lots of soluble cues and factors that influence
00:00:42.01 their behavior in really dramatic and important ways.
00:00:45.13 And one really important type of informational cue comes from the information
00:00:55.10 that cells get from interacting with the extracellular matrix.
00:01:00.01 Extracellular matrix interaction occurs at specialized zones of the cell surface,
00:01:09.12 which in cultured cells are called focal adhesions.
00:01:12.11 These focal adhesions, as we discussed last time,
00:01:16.28 are areas that are rich in integrin adhesion receptors,
00:01:22.17 and they are playing a really important role
00:01:26.23 in bidirectional transmembrane communication.
00:01:32.21 The focal adhesions sense extracellular matrix,
00:01:36.12 but they also are involved in sensing cues that are present on cell surfaces.
00:01:41.03 And they also, as we will discuss in part three,
00:01:44.12 can sense physical stress such as mechanical stimulation.
00:01:51.22 The responses that cells launch as a result of the signaling through
00:01:58.20 these specialized regions of the membrane are really
00:02:01.13 very diverse and include control of cell growth or cell death,
00:02:05.26 cell motility, cytoskeletal organization,
00:02:09.27 and these signals can even result in changes in
00:02:14.05 gene expression, illustrating that there must be some mechanism by
00:02:18.01 which the molecules present at these adhesion sites
00:02:22.09 are able to communicate with the nucleus.
00:02:25.06 Integrins are the primary receptor for extracellular matrix
00:02:30.22 that are located at these specialized adhesion zones.
00:02:36.11 They are concentrated at these focal adhesions.
00:02:39.11 They mediate transmembrane bidirectional
00:02:41.29 signaling, and a really interesting challenge has been to try and understand
00:02:47.01 how they signal to effect so many really important cell behaviors.
00:02:52.08 Because unlike growth factor receptors,
00:02:54.22 they themselves don't have any intrinsic catalytic activity.
00:02:58.18 Rather they seem to operate by recruitment of a large collection of
00:03:04.11 cytoplasmic proteins to the cytoplasmic face
00:03:11.04 of the plasma membrane, and it is these proteins
00:03:13.27 which really facilitate integrin signaling function.
00:03:18.02 There are over 50 components that are present
00:03:22.04 with integrins at these focal adhesions,
00:03:25.28 and these are highly dynamic structures where proteins
00:03:30.01 are coming and going. There are both structural components
00:03:33.23 and catalytic constituents. Here you can see an example
00:03:37.09 of the evidence of one catalytic constituent, and that
00:03:42.03 is tyrosine kinases and their substrates
00:03:45.28 because here we are labeling the cell with an antibody
00:03:48.26 directed against phosphotyrosine.
00:03:50.29 And you can see that these bright patches here
00:03:55.25 where phosphotyrosine is accumulated represent
00:03:58.26 the focal adhesions where integrins would also be concentrated
00:04:03.10 and where you can see there is a very close connection with the ends
00:04:07.22 of the actin filaments, the stress fibers that terminate at these sites.
00:04:13.14 One protein that is one of the many proteins present at these focal adhesions
00:04:20.11 is a protein that was discovered in my laboratory,
00:04:23.02 several years ago, called zyxin.
00:04:25.15 This was a really interesting discovery process, I think,
00:04:31.03 and one that was very serendipitous.
00:04:33.01 In fact, I was making a lot of different antibodies against focal adhesion
00:04:38.05 constituents. At that time we only knew of three focal adhesion constituents,
00:04:42.15 and I was trying to look for new ones.
00:04:44.02 And I was making antibodies against some candidate proteins
00:04:47.24 and one of the rabbits, prior to immunization
00:04:50.12 actually was producing an antibody that already stained
00:04:55.04 focal adhesions of cultured cells.
00:04:56.22 And so I followed what this antibody was recognizing,
00:05:02.07 and it turned out it was recognizing a protein that was
00:05:05.04 previously not appreciated as a focal adhesion constituent.
00:05:07.29 And this protein now has the name zyxin,
00:05:12.04 which comes from the Greek root zeugos, which means to join together,
00:05:17.14 because it is at places where actin filaments are joined to the plasma membrane.
00:05:20.21 This is a very interesting protein that had a number of
00:05:25.20 unusual structural features: highly enriched in proline residues
00:05:30.12 and it also had 3 C-terminal double zinc finger motifs called
00:05:36.16 the LIM domain. The LIM domain was first identified in three transcription factors
00:05:42.25 Lin-11, Isl-1 and Mec-3. And these are all homeodomain proteins
00:05:49.11 that contained two N-terminal LIM domains.
00:05:54.14 In collaboration with Mike Summers and Dennis Winge
00:05:57.17 we solved the structure of the LIM domain,
00:05:59.19 and we determined that it is really a double zinc finger structure
00:06:04.10 with two sort of independent modules, each of which binds a zinc atom.
00:06:10.24 And surprisingly, although many zinc fingers are involved in direct
00:06:17.24 nucleic acid binding, it's quite clear from our analysis that
00:06:22.13 LIM domains serve as protein binding interfaces.
00:06:26.03 However, I should point out that this structure of
00:06:30.09 the LIM domain is very, very similar to the GATA-1 DNA binding domain,
00:06:35.09 so there remains a possibility that in addition to protein-protein interaction,
00:06:39.17 LIM domains may be able to associate with nucleic acids,
00:06:43.05 although this has not been demonstrated directly.
00:06:46.28 There are now a number of LIM domain proteins that have been identified-
00:06:52.12 more than 50 human proteins, and they are
00:06:57.04 functionally quite diverse. Some of them have
00:07:00.00 catalytic features, such as serving as kinases,
00:07:04.00 others are present exclusively in the nucleus and are known
00:07:08.01 to play a role in regulation of gene expression
00:07:11.28 and many others are found in association with the cytoplasm.
00:07:16.20 And what I'll tell you today is that this protein zyxin,
00:07:20.11 seems to be a multifunctional protein that resides
00:07:24.00 prominently at the focal adhesions, but also
00:07:26.27 has... where it seems to regulate cell motility
00:07:30.10 and perhaps even cell death, but it also has
00:07:36.05 the capacity to move into the nucleus, so it is an amazingly interesting protein
00:07:41.02 that is a candidate for participating in direct communication
00:07:44.21 between the cell surface and the nuclear compartment.
00:07:48.28 Zyxin also, we now have come to appreciate,
00:07:55.04 may be implicated in a particular type of cancer: Ewing's sarcoma.
00:08:00.11 I want to tell you a little bit about Ewing's sarcoma, and then
00:08:04.22 about the connections between this focal adhesion protein
00:08:08.28 and its properties and the progression
00:08:11.09 of Ewing's sarcoma that we now understand from pre-clinical model studies.
00:08:17.16 Ewing's sarcoma is a very devastating childhood and young adolescent
00:08:24.26 tumor. It's a small, round, blue cell tumor
00:08:28.22 that you can see histologically is very homogeneous
00:08:33.18 in terms of the way it appears at the cellular level.
00:08:37.23 It's a tumor of the bone, extremely rare, but very, very aggressive.
00:08:43.24 And although it is rare, because it impacts children and young people
00:08:48.13 there's been a fair amount of emphasis on trying to understand its cause
00:08:54.08 and develop better treatment strategies.
00:08:56.21 Right now, we are faced with the challenge that metastatic
00:09:01.01 Ewing's sarcoma is really a lethal disease.
00:09:05.12 Here you can see some data looking at survival
00:09:08.22 from Ewing's sarcoma if the disease was localized,
00:09:15.23 where there's a really good, greater than 80% survival rate,
00:09:20.13 versus if the tumor has spread.
00:09:25.23 And many, many cases of Ewing's sarcoma have micro-metastases
00:09:28.25 at the time of initial detection, and
00:09:31.02 you can see that once one has metastatic disease
00:09:35.04 there is a very, very poor prognosis.
00:09:38.00 Even more strikingly, I think is the fact that we really don't have effective therapies
00:09:45.06 to treat this disease. And here you can see
00:09:47.11 the outcomes for patients after relapse from their initial
00:09:54.00 disease, and those treated with the most aggressive therapy really
00:10:00.18 ultimately do not do much better than those that receive palliative care.
00:10:05.05 So unfortunately this is although a rare tumor, an extremely aggressive and
00:10:11.08 difficult to treat tumor. We understand now that
00:10:16.10 Ewing's sarcoma is caused by a reciprocal chromosome translocation,
00:10:21.00 and 11;22 translocation that results in the productive fusion of
00:10:26.13 a region of the EWS gene with a region of the FLI1 gene.
00:10:32.17 And here you can see one gets out of that fusion a chimeric protein,
00:10:39.00 the EWS-FLI1 chimera, which brings together a transactivation domain from EWS with
00:10:46.05 a DNA binding domain from FLI1.
00:10:50.17 This protein is believed to function as an aberrant
00:10:54.20 transcription factor that we know is responsible for
00:11:00.19 causation of Ewing's sarcoma.
00:11:02.21 In the lab we can demonstrate that EWS-FLI1 expression is sufficient to behave
00:11:11.17 as an oncoprotein, and it's sufficient to transform cells.
00:11:14.26 Here are on the left you can see cells grown in soft agar
00:11:19.10 when they are expressing FLI1, and you can't really see any colonies developing here.
00:11:23.26 But when those cells are expressing the EWS-FLI1 fusion protein,
00:11:30.27 you can see many colonies growing
00:11:34.16 in this soft agar cloning assay.
00:11:37.19 So these cells display anchorage independent cell growth
00:11:41.27 and behave as transformed cells by this classical assay.
00:11:47.17 So a number of groups have been really interested in trying to understand
00:11:52.05 what happens within cells when you express EWS-FLI1,
00:11:56.13 and there's been a recent report by Amsellem and colleagues
00:12:01.26 in which they explore what is the difference between gene expression
00:12:07.18 patterns in 3T3 cells and in 3T3 cells that are programmed to express the EWS-FLI1
00:12:15.14 oncoprotein. And their analysis identified several genes which were
00:12:22.14 misregulated or displayed altered expression patterns
00:12:26.21 when EWS-FLI1 was present. And one of these was
00:12:32.10 the focal adhesion protein zyxin that I mentioned
00:12:35.17 was discovered by my laboratory.
00:12:38.10 You can see here if we look at the level of protein in a 3T3 cell control
00:12:44.04 using an anti zyxin antibody, you see
00:12:46.17 this much protein, and a typical 3T3 cell expressing
00:12:51.04 EWS-FLI1 shows this dramatic reduction in the level of the zyxin protein.
00:12:56.27 And here you can see this again by immunocytochemistry,
00:13:00.10 where in the control cells you can see zyxin nicely concentrated
00:13:04.18 at these focal adhesions, whereas in the
00:13:08.26 3T3 cells that have been transformed with EWS-FLI1,
00:13:13.19 there's both an altered morphology and a loss of zyxin at the focal adhesions.
00:13:20.10 Interestingly of course, when you put EWS-FLI1, a transcriptional regulator into cells,
00:13:29.03 you are expecting to have many, many dramatic changes occur
00:13:33.04 within those cells. So seeing that zyxin is down and it is
00:13:35.20 correlated with this change in morphology
00:13:37.10 is one thing, but knowing that it is responsible, and to what extent
00:13:41.24 it might be responsible for that change in morphology
00:13:43.28 is an open question that needs to be addressed directly.
00:13:48.01 And it was addressed by these investigators by taking these
00:13:52.05 3T3 cells that are transformed with EWS-FLI1,
00:13:57.14 which again show this really dramatic altered morphology
00:14:01.08 from this really well spread, fibroblast-like morphology
00:14:07.00 to a much more apparently motile and non-spread morphology.
00:14:17.06 And what they were able to do was to ask, okay,
00:14:19.13 in these cells, if we put back a wildtype zyxin,
00:14:23.11 what happens? And remarkably you can see this really dramatic phenotypic
00:14:29.13 reversion where the simple re-expression of this one gene, the zyxin
00:14:34.21 gene, causes a phenotypic reversion to something that looks
00:14:38.03 much more like the untransformed cells.
00:14:41.11 So this provided an indication that the zyxin protein
00:14:45.22 was not only reduced in expression, and in response to EWS-FLI1
00:14:51.20 transformation, but also might be responsible for some of the
00:14:56.13 transformed phenotypes that is associated with EWS-FLI1 expression.
00:15:02.23 So my laboratory has been interested in trying to understand what is the function of zyxin.
00:15:08.07 And we took an approach of generating a targeted disruption
00:15:12.20 of the zyxin gene in order to completely eliminate all expression of zyxin
00:15:17.27 and then be able to study the consequences of loss of zyxin
00:15:22.01 expression for cell function.
00:15:24.19 And here you can see that we have successfully generated cells
00:15:29.21 that lack zyxin. Here's wildtype cells, mouse embryo fibroblasts,
00:15:34.29 expressing zyxin. You can see nice focal adhesions
00:15:38.01 staining. And over here, you can see... you can't see. There is a cell here.
00:15:42.24 And it has been labeled with anti-zyxin antibody and there is no zyxin
00:15:46.28 protein. And this is confirmed by the Western blot here showing that
00:15:52.03 the targeted gene disruption was effective
00:15:55.04 and completely eliminates the zyxin protein.
00:15:58.06 Weâ€™ve now begun to characterize those cells
00:16:03.01 and trying to think about how the changes in those cells
00:16:06.20 might be relevant for this Ewing's sarcoma
00:16:11.16 phenotype that was observed by Amsellem and colleagues.
00:16:14.27 First thing that we looked at since we knew that zyxin was present at these
00:16:20.13 focal adhesions where actin filaments are tethered to the plasma membrane
00:16:24.05 is whether the actin cytoskeleton was disturbed in the zyxin null cells.
00:16:29.23 And here you can see that although the zyxin null cells
00:16:35.09 display actin stress fibers-so here are control cells
00:16:39.22 and then null cells-both display actin stress fibers, so we can't
00:16:44.15 conclude that zyxin is absolutely essential
00:16:47.24 for the establishment of these actin arrays in cells.
00:16:51.14 But if we perturb this system and challenge it with a Rho kinase inhibitor,
00:16:57.03 and remember now Rho signaling is important for building
00:17:02.07 of robust stress fiber arrays, and Rho acts in part through
00:17:06.21 the activity of the Rho kinase. So if we perturb Rho kinase signaling
00:17:13.12 with a Rho kinase inhibitor- if we treat wildtype cells with a Rho kinase inhibitor for short
00:17:20.06 periods of time under conditions where we don't see dramatic reduction in the stress fibers.
00:17:25.21 If we look at null cells, we see that the stress fibers are completely eliminated
00:17:31.22 when zyxin isn't there. And this would be
00:17:34.08 the ultimate outcome for the wildtype cells too
00:17:38.06 if we continued this treatment for a longer period of time.
00:17:41.11 So these results illustrated that although the actin stress fiber arrays are
00:17:49.10 present in both wildtype and zyxin null cells
00:17:52.14 that the arrays that are present in the zyxin null cells are less robust
00:17:57.02 and are more sensitive when stress fiber pathways are compromised.
00:18:03.10 And this is actually very interesting because there is a long history illustrating that
00:18:11.18 stress fiber content is inversely related to motility. That is that
00:18:15.13 the bigger and more robust the stress fibers are
00:18:19.05 the less motile the cells are. So you can imagine then that
00:18:23.19 if the zyxin null cells have reduced stress fiber content
00:18:28.29 or less robust stress fiber content
00:18:31.12 that one might anticipate that the cells would show enhanced motility.
00:18:36.05 And indeed that is exactly what we see. We've looked at
00:18:39.24 the zyxin null cells and evaluated their motility properties,
00:18:43.22 in a number of different ways. And here is just one experiment
00:18:47.12 where we looked in a monolayer wound assay.
00:18:51.17 We took wildtype and zyxin null cells, grew them
00:18:56.17 in a monolayer, and then scratched a wound in that monolayer,
00:19:01.16 measured that wound gap, and then monitored the recovery of that wound as the cells
00:19:09.29 migrated into the cleared area.
00:19:12.13 And this is just an endpoint analysis shown here,
00:19:16.11 but after eight hours you can see that the zyxin null...the wildtype
00:19:19.28 cells still have a pretty substantial gap in this wound,
00:19:23.05 whereas the null cells show complete recovery of the wound.
00:19:29.09 And a blind analysis of a large number of wounding assays
00:19:34.15 and quantitation of the velocity of cell migration revealed that
00:19:38.04 the zyxin null cells actually migrate about twice as fast as the wildtype cells,
00:19:46.26 consistent with the idea that the loss of zyxin could be involved in tumor progression
00:19:55.08 and motility. Zyxin null cells also seem to display migratory properties that are
00:20:02.11 uncoupled from extracellular matrix cues.
00:20:05.05 We talked a lot in the first part about how cells normally get
00:20:09.17 substantial signals from their environment and how that has an impact on
00:20:14.16 the signaling output and the behavior of the cells.
00:20:17.14 And in this experiment in which we've examined and compared
00:20:22.21 wildtype cells and zyxin null cells
00:20:24.08 for their migration properties in a Boyden chamber, a transwell migration assay,
00:20:30.27 we have some indication that the zyxin null cells are migrating
00:20:35.26 at sort of maximum speed regardless of any extracellular matrix cues.
00:20:41.23 And this is a really striking phenotype.
00:20:44.27 Here you can see in the open bars the wildtype
00:20:47.28 cells and what we've done here is to put cells on the top of this Boyden chamber
00:20:53.26 and then put different amounts of extracellular matrix protein, in this case fibronectin,
00:21:00.24 on the underside of that filter and allowed the cells to migrate through.
00:21:04.20 And what you can see is that wildtype cells don't migrate very well at all
00:21:10.00 when there is no fibronectin present, no sort of signal,
00:21:13.09 nothing to grab onto on the other side of that filter.
00:21:17.19 And as the fibronectin level increases, you can see that the
00:21:22.18 wildtype cells begin to migrate effectively
00:21:26.11 through the pores and onto the other side of the filter.
00:21:29.29 Interestingly the null cells in comparison, start out, even
00:21:34.25 with no extracellular matrix cues, migrating at maximal velocity
00:21:39.28 and seem unaffected by increasing fibronectin concentration.
00:21:45.25 So what this suggests is again consistently showing us
00:21:50.03 that the zyxin null cells are migrating more effectively than wildtype cells.
00:21:54.02 But also suggests that in the absence of zyxin
00:21:57.25 the cells are sort of almost primed for migration.
00:22:01.04 They appear to be behaving as if they have a signal
00:22:04.19 that there was matrix present when there really isn't any matrix present.
00:22:08.13 Now going back to the Ewing's sarcoma model system then,
00:22:13.13 what is the impact of the expression of EWS-FLI1 oncoprotein
00:22:21.05 in 3T3 cells vis-Ã -vis migration?
00:22:24.14 And does restoration of zyxin expression also suppress a migratory phenotype?
00:22:33.01 So here you can see work again from Amsellem and colleagues
00:22:37.05 where we are looking at migration of normal 3T3 cells
00:22:42.09 following sort of tracks of cells and measuring
00:22:46.08 net displacement. And here you can see when we put in
00:22:51.04 the EWS-FLI1 transgene into those cells in the blue bars,
00:22:56.23 we see a dramatic increase in motility,
00:23:00.27 consistent with what you'd expect for a transformed cell.
00:23:05.06 And interestingly... and we see down here
00:23:10.07 that zyxin levels have fallen there as I showed you before.
00:23:15.18 Now interestingly when you put zyxin back into these cells
00:23:19.10 what are the consequences for cell motility?
00:23:21.06 Well, you see a restoration of the more slow, wildtype motility.
00:23:27.06 So once again it appears that the changes in zyxin levels
00:23:32.27 are contributing very substantially to the motile phenotype of these cells.
00:23:38.06 And I think that this...some insight into how this
00:23:42.08 occurs may come from work of Yu-li Wang's lab, where he followed individual cells
00:23:50.07 that were plated on a polyacrylamide substratum
00:23:55.05 into which he embedded fluorescent latex beads
00:23:57.29 so he could really look at how much force
00:24:00.19 is generated at various adhesion points,
00:24:04.08 tethering points between the cell and the substratum.
00:24:10.23 And he noticed a very interesting thing, and that is that when you
00:24:17.03 compare the traction stress maps, where they are really measuring
00:24:20.21 the deflection of these beads with the location and intensity
00:24:24.28 of expression and localization of a GFP-zyxin,
00:24:29.24 what they observed is that where you have really, really high traction forces
00:24:35.11 at this leading edge here shown in this deeper red color,
00:24:38.00 you see that the area is really devoid of zyxin.
00:24:42.01 So, and then you can follow over time a single, the development of a single
00:24:48.18 focal adhesion and see that this focal adhesion starts out
00:24:54.05 with no zyxin and then the zyxin level goes up, up, up
00:24:58.14 and then plateaus. And strikingly, as the zyxin levels are going up here
00:25:03.13 the traction stress is going down.
00:25:06.20 So clearly there is an inverse relationship between
00:25:09.15 the presence of zyxin at these focal adhesions
00:25:12.21 and traction stress, and this could be a very interesting functional explanation
00:25:19.11 for why the cells that lack zyxin are more migratory,
00:25:24.25 that is they have the capacity to generate a greater or more sustained
00:25:29.05 traction stress. And that is something that we're all very interested in looking at
00:25:33.01 in more detail in the future.
00:25:35.00 So in summary of this part of the presentation then,
00:25:38.03 I described the Ewing's sarcoma as a lethal, devastating disease
00:25:46.02 of childhood, fortunately rare which is caused by
00:25:51.14 a reciprocal translocation that gives rise to the EWS-FLI1 oncoprotein.
00:25:57.05 EWS-FLI1 expression in a model Ewing's sarcoma system
00:26:03.18 results in decreased zyxin, and our knockout studies
00:26:08.22 showed that actin stress fibers are less robust in zyxin null cells,
00:26:13.11 and that decreased zyxin is associated with enhanced cell motility.
00:26:17.23 So how is this enhanced motility and impact on the actin cytoskeleton achieved?
00:26:26.02 Well, we learned something about this actually
00:26:29.19 by a sort of surprising link by studying Listeria,
00:26:35.02 an intracellular pathogen that is really the subject of Julie Theriot's
00:26:39.12 iBioSeminar presentation, so you can learn much more about
00:26:43.26 Listeria by looking at her presentation.
00:26:46.00 But, studies of Listeria provided some important insight into zyxin function
00:26:53.20 and its mechanism of action.
00:26:54.23 Just briefly, Listeria is an intracellular bacterium
00:27:00.08 that invades its host and is taken up by phagocytosis.
00:27:04.12 It's a foodborne bacterium that can cause really
00:27:09.29 devastating disease, particularly in immunocompromised individuals.
00:27:13.25 But the cell biology of this organism is that it
00:27:16.25 gets into these phagosomes and manages to
00:27:20.22 escape from this phagosomal compartment
00:27:23.18 and gets released into the cytoplasm where it can replicate. And for the
00:27:27.08 purposes of our discussion, it harnesses
00:27:29.17 the host cell machinery for actin assembly,
00:27:32.02 growing this nice long actin comet tail,
00:27:36.23 which allows it to move within the host cell and actually generate these
00:27:43.07 microspike projections which enable it to invade neighboring cells.
00:27:47.21 And in this way, it completely is able to transmit itself from one
00:27:51.14 cell to the next without ever leaving the protection of the intracellular
00:27:56.25 environment. And from a variety of investigations
00:28:02.02 we now understand that there is a single protein present on the Listeria surface,
00:28:07.09 the ActA protein, which is both necessary and sufficient for pathogenicity of this bacterium.
00:28:13.22 And it's necessary and sufficient for the ability of these bacteria to generate
00:28:20.11 these actin comet tails and thus move within the host cell and be transmitted
00:28:26.03 from one cell to the next. The ActA protein
00:28:31.00 is a protein that is anchored in the bacterial surface via a membrane anchor
00:28:36.18 and then has a long protein sequence area which
00:28:43.07 is present in the host cytoplasm.
00:28:45.29 And genetic analysis via mutational studies and deletion studies
00:28:51.03 have shown that there are two really critical functional domains in ActA,
00:28:55.18 which are important for its ability to cooperate with host cell machinery to build
00:29:02.09 this actin comet tail on the surface of the bacterium.
00:29:06.02 One is the domain here which is critical for the nucleation of actin assembly.
00:29:09.21 And the other is a series of proline repeats here which are critical for acceleration
00:29:15.04 of actin assembly on the bacterial surface.
00:29:18.06 And so, in collaboration with Daniel Louvard's lab, we made a bunch of
00:29:23.09 antibodies against Listeria ActA with the idea of trying to look for what endogenous cellular
00:29:29.02 proteins ActA might be mimicking on the surface of the bacterium, in order to
00:29:34.24 recruit host machinery and stimulate the elaboration of this actin comet tail.
00:29:39.25 And when we did this we had the surprising result
00:29:45.18 that antibodies against Listeria ActA recognized
00:29:49.03 just a single eukaryotic protein, a single human protein,
00:29:53.27 at least in our experiments. And that protein turned out to be zyxin.
00:29:57.27 And interestingly, it turns out that
00:30:01.01 this really important proline rich domain in ActA,
00:30:04.01 which is critical for acceleration of actin assembly,
00:30:06.15 is almost completely conserved in zyxin,
00:30:10.10 giving rise to the idea that zyxin might be able to stimulate actin assembly.
00:30:16.07 And indeed zyxin shares with ActA the ability
00:30:20.07 to interact with proteins in the Ena/VASP family,
00:30:23.08 which are critical regulators of actin assembly
00:30:27.19 that recruit profilin and actin and by multiple mechanisms
00:30:32.26 enhance actin assembly. So, returning back to this Ewing's sarcoma model then,
00:30:41.13 we see that in model Ewing's sarcoma cells that are expressing the EWS-FLI1
00:30:47.03 transgene, that's associated with loss of zyxin,
00:30:54.14 decreased actin stress fibers, which are associated with loss of zyxin,
00:30:58.15 as is increased motility. And so, I think it's
00:31:03.16 going to be extremely interesting to explore further
00:31:06.21 what is the role of zyxin in human patient
00:31:10.10 samples, and evaluate whether or not loss of zyxin
00:31:14.23 is correlated with more aggressive disease.
00:31:18.05 Interestingly, in the original Amsellem experiments
00:31:23.10 they also, in addition to looking at motility,
00:31:26.02 looked at whether zyxin has an impact on anchorage independent
00:31:31.25 cell growth of this EWS-FLI1 transformed fibroblasts.
00:31:35.19 And they found that it had really quite a dramatic impact.
00:31:40.11 So here you can see again normal 3T3 cells
00:31:43.26 that are not transformed don't grow in soft agar,
00:31:49.06 and so you don't have any colonies.
00:31:51.21 If you express, if you transform those cells with a control
00:31:57.19 vector, you don't see any change.
00:32:00.15 If you express more zyxin in those cells you don't see any change.
00:32:04.08 But look, as we saw in the original soft agar cloning experiment that I showed you
00:32:09.11 earlier, when you express EWS-FLI1 in those 3T3 cells, you see
00:32:15.12 a dramatic increase in the ability of those cells to grow in
00:32:19.14 soft agar. And you can suppress that ability by re-introducing zyxin into those
00:32:25.19 cells. So this was, in addition to the motility
00:32:29.18 phenotype, I think a very striking piece of data that
00:32:33.28 suggested that zyxin might also be influencing
00:32:37.21 either cell proliferation or apoptosis and contributing to this suppression
00:32:45.07 of anchorage independent cell growth.
00:32:48.15 Interestingly, I told you that it is very, very difficult to
00:32:54.11 treat Ewing's sarcoma, so there are many
00:32:56.11 many investigators really working hard to identify new strategies
00:33:01.17 for therapeutic intervention with this disease.
00:33:05.26 And one interesting new concept that several groups are working on
00:33:10.29 is the use of an antibody directed against a cell
00:33:15.21 surface component, CD99, which is highly expressed
00:33:19.29 on Ewing's sarcoma cells, human tumor cells.
00:33:24.21 And it turns out that if you take this anti CD99 antibody and
00:33:29.14 expose Ewing's sarcoma cells to this antibody, the clustering of that
00:33:36.10 cell surface marker somehow induces apoptosis of the Ewing's sarcoma cells
00:33:43.03 by a mechanism that at this point in time really isn't understood.
00:33:46.20 I mean we really don't understand yet what CD99's normal function is.
00:33:50.08 But because it is so highly enriched on Ewing's sarcoma cells
00:33:53.19 and because engagement of CD99 causes apoptosis, it's
00:33:58.25 really being thought of as a potential therapeutic intervention for Ewing's sarcoma.
00:34:05.10 And interestingly, in the course of trying to understand
00:34:09.18 how clustering and engagement of anti-CD99
00:34:13.23 with anti-CD99 antibodies causes apoptosis.
00:34:18.14 investigators found that zyxin is a critical componentin that apoptotic pathway.
00:34:27.21 And here you can see some flow cytometry analysis looking at
00:34:33.03 the ratio of viable cells to apoptotic or necrotic cells
00:34:36.15 before or after anti-CD99 treatment, and
00:34:44.14 with normal zyxin levels or suppressed zyxin levels.
00:34:48.18 And here you can see that in the normal cell population
00:34:53.06 76% of the cells are in the viable channel here.
00:35:00.01 And if you engage those cells with the anti-CD99 monoclonal antibody,
00:35:08.24 which is called 0662, you can see a shift from the viable channel to
00:35:14.26 the apoptotic channel consistent with the idea that
00:35:19.15 the monoclonal antibody can induce apoptotic signaling
00:35:22.23 in these cells. Now that doesn't shift at all when
00:35:29.04 you use a control antisense construct,
00:35:33.12 but if you use a construct that is specifically designed to knock down zyxin levels,
00:35:38.04 what you see is that you have a reduction in the apoptotic response
00:35:46.16 suggesting that zyxin is at least partially involved or partially required
00:35:52.00 for the ability of anti-CD99 to induce apoptosis.
00:35:56.09 And so, this leads to an interesting addition to the
00:36:04.03 prior thinking about zyxin function that in addition to
00:36:08.17 contributing to regulation of motility,
00:36:12.28 that it may actually be a pro-apoptotic factor under some circumstances
00:36:17.29 and loss of zyxin might contribute to increased survival.
00:36:21.16 So this is quite interesting in the sense that
00:36:24.06 we know that expression of EWS-FLI1 causes reduction in zyxin expression in this
00:36:30.26 3T3 model, and that would be then associated with
00:36:34.06 increased motility and increased survival based on the cell based studies.
00:36:39.26 And so I think a very important future goal is to really now
00:36:44.07 go and test whether zyxin expression level is an indicator of tumor stage
00:36:50.23 and in addition, whether or not it will predict therapeutic response.
00:36:56.18 For example if zyxin levels are extremely low,
00:36:59.22 that might give us the insight that those tumors
00:37:04.08 have greater metastatic potential because
00:37:08.07 of the increased motility associated with loss of zyxin.
00:37:10.29 Interestingly, many investigators are now starting to find zyxin down regulated
00:37:18.08 in a variety of tumors, and here's an example showing that it is down regulated
00:37:24.12 in invasive bladder cancer. Here's the superficial tumor where the brown staining
00:37:29.18 indicates there is still robust zyxin expression.
00:37:32.11 And in this invasive carcinoma you see
00:37:35.09 very little expression of zyxin, if any at all.
00:37:38.15 And these investigators did a very extensive analysis to look
00:37:42.26 at association of a number of potential biomarkers
00:37:48.14 with tumor stage and grade and found that expression levels for zyxin,
00:37:54.02 as well as a couple of other markers, were directly associated with tumor stage and grade.
00:37:59.09 So, this could be another important and valuable
00:38:03.18 insight that allows clinicians to be able to identify tumors
00:38:08.28 and diagnose them with a higher degree of resolution using
00:38:12.28 these types of biomarkers to further refine
00:38:16.06 the grade of the tumor. Now I mentioned in the beginning that focal adhesions also are
00:38:22.11 really important for communicating with the cell nucleus,
00:38:25.24 and that these signals that emanate from focal adhesions
00:38:30.26 also influence gene expression. And I think one of the biggest challenges
00:38:34.22 that we face as cell biologists is
00:38:38.04 knowing that that occurs, not really understanding what is the mechanism
00:38:42.13 of action by which these events that are happening at the cell surface
00:38:47.05 are communicated to the nucleus to directly regulate
00:38:51.01 gene expression. And I am sure there are going to be a number of mechanisms
00:38:57.01 involved here, but one thing that I like to point out is that the zyxin protein
00:39:02.25 may be a candidate for communicating with the cell nucleus.
00:39:07.00 And indeed a student in my lab recognized a number of years ago
00:39:11.12 that the zyxin protein has sequence that looks very similar to
00:39:16.16 a nuclear export signal, showing these very highly ordered leucine residues
00:39:22.11 that are in similar positions to nuclear export signals
00:39:26.13 in the Rev protein of HIV1 as well as IkB.
00:39:31.17 So we looked to see whether if we deleted that leucine rich region of zyxin if it
00:39:37.16 had an impact on the subcellular distribution of zyxin,
00:39:41.10 and here you can see expression of wildtype zyxin in cells. And at steady state
00:39:46.18 you really see the zyxin concentrated in these focal adhesions,
00:39:50.03 and very little zyxin if any detectable by
00:39:52.13 immunocytochemistry within the nucleus.
00:39:54.11 But, if we delete just these 17 amino acids from zyxin, this leucine rich region,
00:40:00.06 and now ask where is that protein concentrated,
00:40:03.23 what we see is this really dramatic shift in the subcellular distribution of zyxin with
00:40:09.07 the protein migrating really substantially from these focal adhesions and accumulating
00:40:14.28 in the nuclear compartment.
00:40:17.26 So to test further whether or not this
00:40:22.06 sequence of zyxin really functions as a nuclear export signal,
00:40:27.05 we did a classic experiment where we took that zyxin sequence
00:40:31.26 and hooked it onto a carrier protein, in this case I am showing you
00:40:36.28 here GST, glutathione S-transferase, and asked
00:40:42.03 whether or not if we inject normal GST or GST tagged with this zyxin sequence
00:40:49.07 directly into cell nuclei, whether the zyxin sequence will support
00:40:54.06 the nuclear export of that GST protein.
00:40:57.27 GST is too large to diffuse through nuclear pores, so if you inject
00:41:02.06 it into the nucleus in the absence of a functional nuclear export signal,
00:41:06.00 it will be retained in the nucleus.
00:41:07.17 And that's exactly what you see with the unmodified GST.
00:41:12.10 You microinject it into the nucleus, and this is a fluorescently tagged version of GST,
00:41:17.05 put it into the nucleus, and it stays there.
00:41:20.09 But if you take just, in this case, amino acids 319 to 335 of zyxin
00:41:26.07 and attach that to the GST through genetic engineering,
00:41:31.05 express that protein, label it, and inject that protein into the nucleus,
00:41:34.29 you see that very, very rapidly that protein winds up in the cytoplasm,
00:41:41.09 illustrating that that short sequence of zyxin has the capacity
00:41:45.03 to serve as a nuclear export signal
00:41:46.26 and transport GST from the nucleus to the cytoplasm.
00:41:50.24 So, this was very exciting because it really suggested that the zyxin protein had a nuclear export signal
00:42:00.21 and perhaps was present in both the focal adhesions
00:42:04.29 and the nuclear compartment and could be part of the
00:42:07.18 machinery that really functionally linked the cell surfaceto the nucleus.
00:42:12.15 However, of course, when you are cutting pieces of proteins up
00:42:15.14 and putting them onto other proteins, things can happen that are unanticipated,
00:42:20.21 and so we really wanted to have a direct mechanism to look at whether zyxin ever
00:42:26.14 goes into the nucleus. And this was also important
00:42:28.20 because, as I pointed out, at steady state we really don't see zyxin
00:42:31.25 in the nucleus, so we developed an assay to report
00:42:36.17 whether zyxin goes into the nucleus.
00:42:39.05 And, I think, this is an experiment that David Nix in my lab
00:42:43.08 did when he was a graduate student,
00:42:44.12 and I think it is really amazing that this experiment worked.
00:42:47.25 He decided to test whether or not zyxin goes into the nucleus
00:42:53.21 by injecting a cocktail of two different antibodies
00:42:57.17 into, directly into the nuclei of individual cells.
00:43:02.07 And the two antibodies were an anti-zyxin monoclonal antibody
00:43:06.23 and a matched control antibody that didn't recognize any cellular proteins.
00:43:11.25 And once again these antibodies, which were labeled with different color fluorochromes,
00:43:18.00 are too large to diffuse out through nuclear pores.
00:43:22.00 So you would expect them to be retained in the
00:43:23.28 nucleus unless they are specifically exported
00:43:27.21 by binding to another protein partner.
00:43:31.08 So here we start out, we inject these two antibodies into cell nuclei.
00:43:36.24 Zyxin is, as I mentioned, concentrated in the cytoplasm,
00:43:39.20 and if zyxin didn't shuttle into the nucleus, you would expect
00:43:44.22 over time for the antibodies to remain trapped in the nucleus
00:43:48.02 and for the zyxin to remain in the cytoplasm.
00:43:51.23 In contrast, if there is shuttling going on and zyxin
00:43:55.22 is moving from the focal adhesions into the nucleus and back
00:43:59.26 out again, then we might expect one of two things.
00:44:03.08 We might expect zyxin to itself become trapped in the nucleus
00:44:09.09 over time along with its antibody partner.
00:44:13.05 Or, alternatively, we might imagine that zyxin could migrate into the nucleus,
00:44:18.17 bind the specific anti-zyxin antibody,
00:44:21.22 and because the zyxin harbors a nuclear export signal,
00:44:25.17 it could actually extract that zyxin partner from the nucleus
00:44:31.25 and facilitate its nuclear export. And surprisingly,
00:44:35.16 this is exactly what we saw.
00:44:38.17 So here you can see a cell into which we have injected
00:44:43.07 control antibody in A and anti-zyxin antibody together into the nucleus of the cell
00:44:49.24 in B. And this is a very early time point after injection, and you can see
00:44:54.06 that both antibodies are prominently concentrated within
00:44:58.07 the nucleus of the cell.
00:45:00.08 And if we wait over time, and then look, what we see (and this is a different cell, of course)
00:45:05.26 the control antibody marks the site of injection. It is retained in the nucleus.
00:45:11.28 But look over here, the anti-zyxin antibody which was co-injected into the nucleus of the cell
00:45:18.18 is completely missing from this nucleus
00:45:21.05 and has now begun to populate these focal adhesions.
00:45:25.17 So this was a very, very striking result
00:45:28.13 which illustrated to us that protein that was in the cytoplasm,
00:45:32.06 zyxin protein in the cytoplasm could move into the nucleus.
00:45:35.08 And that protein once dwelling in the nucleus
00:45:38.22 could come out of the nucleus and go back to the focal adhesions,
00:45:42.15 really, clearly suggesting a mechanism for communication
00:45:46.15 between these two cellular compartments.
00:45:49.17 And we can now show using leptomycin, an agent that inhibits
00:45:56.15 nuclear export, that indeedif we treat cells with leptomycin to inhibit
00:46:02.01 nuclear export that zyxin begins to accumulate in the nuclear compartment.
00:46:08.25 And if we look at large populations of cells,
00:46:10.16 we see this occurs in an asynchronous fashion
00:46:13.16 illustrating that there must be some physiological signals
00:46:16.17 that stimulate the release of zyxin from the focal adhesions
00:46:21.24 and the import of the protein into the nucleus.
00:46:25.21 And of course, a really important unanswered question
00:46:29.26 is what those signals might be.
00:46:32.26 So what could be the functional significance of nuclear shuttling of zyxin?
00:46:39.00 Well, I think there are two general possibilities. One is that the zyxin protein
00:46:43.20 could itself serve as a nuclear factor. You could imagine that the zyxin
00:46:48.15 protein is sort of retained at focal adhesions
00:46:51.14 where it's awaiting some sort of signal
00:46:55.11 or information from the extracellular environment
00:46:59.02 and under certain conditions would be released,
00:47:02.20 allowed to move into the nuclear compartment, and perhaps even
00:47:06.05 participate directly in regulating gene expression or some other nuclear function.
00:47:11.00 And this is a really intriguing idea, although to date
00:47:18.16 there's not really any direct evidence for a role for zyxin
00:47:24.14 within the nucleus, and particularly not in regulation of gene expression.
00:47:29.19 Though, I'll point out that the zyxin protein, remind you that the zyxin protein
00:47:34.16 has these LIM domains, which are found also in many transcriptional regulators,
00:47:40.22 so there remains, I think, an intriguing possibility that zyxin
00:47:44.09 could be directly influencing some nuclear activity.
00:47:47.21 The other alternative is a more general one that suggests that zyxin is, again,
00:47:55.10 sitting at these focal adhesions here, and then in response
00:48:00.18 again, to some signal, perhaps it could move into the nucleus
00:48:04.10 and recover a protein out of the nucleus,
00:48:09.13 returning it to the cytoplasm, and thus downregulating
00:48:11.28 the activity of that factor. Or alternatively
00:48:15.01 it could be a chaperone that really is designed
00:48:20.24 to retain a nuclear factor in the cytoplasm.
00:48:24.24 So for example, zyxin could be sitting at the focal adhesion
00:48:28.20 holding onto the protein that has a nuclear localization signal.
00:48:31.17 When that protein is bound to zyxin, if it ever went into the nucleus it would be carried
00:48:36.07 rapidly out because of the NES on zyxin.
00:48:38.11 But under certain conditions you could imagine that that protein would be
00:48:42.09 specifically released from zyxin, allowed to move into the nucleus
00:48:46.07 to activate some nuclear function.
00:48:48.29 So again, we now understand that this zyxin protein
00:48:52.15 clearly shuttles between the focal adhesions,
00:48:54.19 these really important signaling zones that are sensitive to extracellular cues.
00:48:59.22 It shuttles between those zones and the nuclear compartment.
00:49:04.27 And I think the next challenge is to try and identify what the
00:49:11.00 specific signals are that stimulate the release of zyxin
00:49:16.09 into the nucleus, and what its specific function there might be.
00:49:20.00 Interestingly, it is now appreciated, just in the last
00:49:24.10 several years, that many LIM domain proteins
00:49:27.19 have this very interesting sort of duality
00:49:31.14 in terms of their subcellular distribution.
00:49:33.24 All of these proteins listed here are proteins that reside both on
00:49:41.08 the actin cytoskeleton or in the focal adhesions, and also have
00:49:45.24 the capacity to move into the nucleus.
00:49:48.03 So I think there's a class of these molecules
00:49:50.25 that are going to be playing a really interesting
00:49:53.17 role in regulation of nuclear function
00:49:56.13 and in communication between the cytoskeleton and
00:50:00.14 the nucleus, real candidates for intracellular communication.
00:50:03.26 So I'll close there and just acknowledge some of the people in my lab
00:50:07.17 that participated in the work I described.
00:50:11.08 I think it's a very exciting time in which we are appreciating
00:50:16.00 much more the important roles of the proteins
00:50:19.24 that are present at these specialized adhesive zones,
00:50:24.09 and beginning to see not only how these proteins
00:50:27.14 influence cell behavior such as motility, perhaps
00:50:31.05 apoptotic signaling, perhaps communication with the nucleus,
00:50:35.22 but also are beginning to be able to put these pathways
00:50:40.24 into a broader context and appreciate how disturbance
00:50:44.17 of the functions of these molecules in these pathways
00:50:47.05 may impact on human disease.

Part 3: Focal Adhesions as Stress Sensors

00:00:03.08 Hello, my name is Mary Beckerle, and I am a professor of Biology
00:00:06.07 and oncological sciences at Huntsman Cancer Institute
00:00:09.28 and the University of Utah. In this last segment,
00:00:13.07 I am going to talk with you about
00:00:14.19 some very interesting and exciting recent results from my laboratory,
00:00:19.08 in which we are trying to understand how cells sense and respond to mechanical cues.
00:00:25.14 We know that cells are really sensitive to a lot of different types of cues,
00:00:35.24 chemical cues, as well as mechanical cues, and what we are trying to understand
00:00:42.19 is how cells maintain a stable environment
00:00:48.01 and how organisms maintain a stable environment
00:00:49.21 in the face of all of this external simulation.
00:00:54.12 Now the concept of homeostasis, or maintaining a stable internal environment,
00:01:01.09 is one that physiologists have been aware of and have been working on
00:01:05.05 for many, many decades.
00:01:07.15 And basically there is in this concept of homeostasis
00:01:12.13 a model cassette for how a stimulus can impact
00:01:19.07 with a sensor and an effector pathway,
00:01:21.24 and then, via a combination of positive and negative feedback,
00:01:26.07 control the environment and maintain stability.
00:01:30.25 So how would this work when we are talking about
00:01:34.16 the exposure of cells and organisms to
00:01:37.19 significant mechanical stress? Well, we know that there are many
00:01:42.26 mechanosensitive organs in our body: bone, muscle, lung, the vasculature,
00:01:50.08 the heart. And if cells and organs don't respond
00:01:55.13 appropriately to mechanical signals, this can lead to very serious consequences
00:02:00.20 and serious disease. In the case of bone, for example,
00:02:05.06 which is a weight bearing organ, if there's not sufficient weight on the bones,
00:02:11.19 this can give rise to disuse osteoporosis.
00:02:15.01 In muscle, as illustrated schematically here,
00:02:19.21 there's a very, very dramatic response
00:02:22.22 to mechanical stimulation, where if muscle is exposed
00:02:27.29 to additional force or weight bearing, this can
00:02:33.14 give rise to significant hypertrophy or expansion of the muscle mass.
00:02:39.17 And in contrast, if muscle is not utilized due to
00:02:46.10 disease or disuse, it can atrophy. And one of the
00:02:51.02 ways in which both hypertrophy and atrophy happen
00:02:54.16 is by expansion of individual cells or the reduction in size of individual cells.
00:03:04.27 So how is it that muscle cells can respond to these mechanical
00:03:10.14 signals, communicate to the nucleus to either
00:03:16.00 activate programs of gene expression that lead to more muscle growth
00:03:19.05 or allow for a decrease in muscle mass when it is not required?
00:03:26.17 Similar response to mechanical stress is evident in the cardiovascular system.
00:03:32.26 Chronic high blood pressure is known to put an additional load on the heart.
00:03:39.01 And when that kind of condition exists,
00:03:43.02 you can see that you get this morphological
00:03:45.07 increase or hypertrophy of the heart.
00:03:48.27 And we now understand that this is not due to the
00:03:52.28 production of additional cardiac cells.
00:03:54.23 We are born with the number of cardiac muscle cells
00:03:57.07 that we are going to have when we die.
00:03:58.12 However, excessive mechanical load or enhanced mechanical load
00:04:03.19 causes the activation of cellular programs that lead to
00:04:09.04 expansion of the size of individual cardiac muscle cells,
00:04:13.27 and this ultimately leads to the expansion of the size of the heart.
00:04:17.17 So here you can see unstimulated or unstressed cardiomyocytes,
00:04:23.18 and here you can see corresponding cardiomyocytes
00:04:27.09 from a heart that has been put under stress.
00:04:32.11 And essentially what has happened here is that those muscle cells
00:04:35.17 are able to sense that increased mechanical load
00:04:39.24 and that signals them to activate and reactivate
00:04:43.12 the fetal program for gene expression
00:04:45.29 so that they build more contractile machinery.
00:04:49.01 This, of course, is initially adaptive and provides
00:04:52.06 for a mechanism by which the cardiac output
00:04:56.15 can be enhanced to compensate for the increased load on the heart,
00:05:03.16 but at some point it becomes maladaptive and leads to cardiomyopathy.
00:05:10.25 So where is the cell's stress sensor and how are these mechanical signals sensed,
00:05:19.11 and how are they responded to?
00:05:22.04 One of the really interesting ideas if you think about a cell
00:05:27.10 that is attached to the extracellular environment,
00:05:30.14 and we have, as we discussed before, these focal adhesions which are specialized
00:05:35.07 regions of the cell membrane that tether
00:05:37.28 the cell to the extracellular environment.
00:05:40.02 You can imagine that if this cell was being stretched,
00:05:43.25 that if you were pulling on this matrix here,
00:05:46.08 that these focal adhesions would really serve appropriately
00:05:50.01 as conduits for transmission
00:05:52.06 of that force or the signals that result
00:05:57.09 from that mechanical stimulation.
00:06:01.08 And so we were really interested in exploring this possibility:
00:06:05.29 first testing what is the normal cellular response to mechanical stretch?
00:06:12.28 One. And where does the stress sensor reside?
00:06:18.21 And three, what are some of the mechanisms by which cells compensate
00:06:23.28 and respond to these mechanical cues?
00:06:26.29 So with Masaaki Yoshigi, my bioengineering colleague,
00:06:31.18 who developed a very, very interesting stretch device,
00:06:36.21 we've taken cells and plated them on an elastic silicone
00:06:44.08 coverslip or sheet, and these cell can...these sheets are then clamped on
00:06:50.07 both ends, and we can expose these cells to cyclic stretch.
00:06:55.22 And we use 60 hertz and about 10 to 15% stretch.
00:07:01.16 And you can see that... this is my movie...
00:07:04.28 and you can see that you can stretch these cells,
00:07:10.07 and now we want to understand what are the consequences
00:07:12.07 of that stretch response.
00:07:15.09 So one of the first things that you see when you stretch cells in this way
00:07:20.28 is a really dramatic change in the actin cytoskeleton.
00:07:25.06 Here is an unstretched cell and you can see
00:07:28.13 the filamentous actin labeled with rhodamine phalloidin,
00:07:32.18 and you can see the actin stress fibers. However, if you stretch
00:07:36.09 these cells, and this particular cell was stretched, I believe, for one hour,
00:07:40.20 you see this really, really dramatic reinforcement of the actin cytoskeleton.
00:07:45.07 The stress fibers are much more robust, much more intense.
00:07:50.20 We developed a mechanism to actually quantitate the stress fiber thickness,
00:07:56.11 and when we do that we can see that even within thirty minutes of stretch
00:08:03.03 we get a really statistically significant enhancement
00:08:06.21 in the thickness of the actin filaments.
00:08:10.03 And this is sustained over a long period of time.
00:08:15.05 Another impact of cyclic uniaxial stretch on cells
00:08:23.27 is a re-orientation, and re-alignment of the actin cytoskeleton
00:08:28.12 perpendicular to the stretch vector. And these are
00:08:31.28 fibroblasts that we are using, and different cells respond in different ways.
00:08:35.15 But very robustly in fibroblasts you start out with
00:08:40.20 unstretched cells that are randomly
00:08:42.21 oriented. And then over time, progressing to about eight hours,
00:08:48.16 you can see that the cells have now begun to align perpendicular
00:08:53.00 to the stretch vector.
00:08:54.01 And again we can quantify this change, and you can see
00:08:57.27 we go from randomly oriented cells with
00:09:03.00 cells oriented in all different directions to
00:09:06.22 a cell population where there is a very high probability that they are going
00:09:11.05 to be aligned perpendicular to the stretch vector within eight hours.
00:09:15.13 So what we see then is with these fibroblast cells we have two
00:09:21.21 really dramatic and consistent responses to mechanical stimulation.
00:09:26.08 We have a cytoskeletal reinforcement, and we have a
00:09:32.17 cytoskeletal re-orientation.
00:09:34.09 And it's really been very unclear how these are related to each other.
00:09:39.11 You could imagine that since the reinforcement occurs
00:09:42.06 on a more rapid time course
00:09:43.29 that it could be required prior to re-orientation,
00:09:48.23 or alternatively in this other model,
00:09:51.29 you could envision that mechanical force independently stimulates
00:09:55.09 these two responses and that they are mechanistically distinct.
00:10:00.21 So we wanted to try and address how
00:10:05.06 the cells are responding to these mechanical cues, and what kinds of
00:10:09.00 changes are happening within the cells that give rise to
00:10:12.13 this cytoskeletal reinforcement and re-orientation.
00:10:16.18 And again, we thought about how the cells are sitting on their
00:10:21.11 extracellular substratum, and essentially when we stretch that
00:10:25.02 silicone membrane, we are pulling on the places where
00:10:30.15 cells are tethered to the matrix, and these are
00:10:32.21 focal adhesions. So we thought it would be very interesting
00:10:35.19 to look and see whether any
00:10:38.10 focal adhesion constituents changed their distribution, or altered it
00:10:42.00 in any way in response to stretch in order to
00:10:44.17 give us some insight into some of the molecular events
00:10:47.16 that occur when cells are exposed to this type of mechanical stimulation.
00:10:52.03 And we really did a survey of a large, large number of focal adhesion
00:10:56.25 constituents to explore their distribution before and
00:11:01.03 after stretch, and I'll just show you two examples
00:11:06.10 here this morning. One is the protein vinculin
00:11:10.22 which is a focal adhesion constituent that does not change
00:11:13.24 its distribution in response to stretch, and then I'll show you
00:11:16.29 a second one, the protein zyxin, which I talked to you about extensively in part two
00:11:22.04 of this series, which is a focal adhesion constituent
00:11:25.27 which really responds quite dramatically and rapidly
00:11:29.13 to mechanical stress. So here you can see
00:11:32.13 in the top two cells here an unstretched cell
00:11:37.22 that's been double labeled with antibodies
00:11:39.16 against vinculin on the left and zyxin over here on the right.
00:11:43.06 And you can see that these two proteins are nicely
00:11:46.26 co-localized at these focal adhesions. After stretch, you can
00:11:52.18 see that the vinculin is still found at these punctate
00:11:56.16 focal adhesions, but the zyxin is basically missing
00:12:00.21 from these focal adhesions and is now populating these long
00:12:03.18 actin stress fibers. And you can see that
00:12:07.27 these two proteins are behaving extremely differently
00:12:11.10 to the stretch stimulus in this overlay image
00:12:14.24 where in the unstretched cells you can see
00:12:19.06 the yellow merge indicating coincidence
00:12:21.21 of the zyxin and vinculin labeling,
00:12:23.25 showing that both proteins are prominently localized
00:12:27.04 at the focal adhesions, whereas in the stretched conditions you see red
00:12:32.09 here where the vinculin is remaining in the focal adhesions,
00:12:37.18 and the zyxin label has now traveled to a new distribution
00:12:41.24 in the cell to the stress fibers.
00:12:44.10 So this was a very, very striking observation
00:12:49.18 that really rapidly upon uniaxial cyclic stretch
00:12:53.21 there's a response within the focal adhesion
00:12:57.07 in which one of the constituents within the focal adhesion
00:13:00.04 rapidly is translocated from the focal adhesions to the actin stress fibers.
00:13:07.18 And we were very interested in this possibility.
00:13:12.11 It was the first example of a protein that was really
00:13:16.01 a mechanosensitive protein that responded in this dramatic way
00:13:21.07 to force, mechanical stimulation. And, as we discussed before,
00:13:27.08 this is the molecular architecture of the zyxin protein,
00:13:31.12 and we of course wanted to know now-we see this dramatic change
00:13:35.15 in zyxin's distribution-is the zyxin protein required
00:13:40.10 for any of the major morphological changes
00:13:43.26 that we see in response to stretch, either the actin reinforcement
00:13:47.17 or the re-orientation of the actin cytoskeleton.
00:13:51.22 So to address this question, we generated a zyxin null mouse,
00:13:57.13 and isolated mouse embryo fibroblasts that completely lack the zyxin protein
00:14:02.28 and then compared the behavior of those zyxin null cells with
00:14:08.03 wildtype cells that are stretched using this uniaxial cyclic stretch mechanism.
00:14:15.10 And here you can see on the left-hand side wildtype cells
00:14:19.25 unstretched and stretched, and just visually I think you can see how striking
00:14:24.23 it is that the cells are oriented in lots of different ways
00:14:28.14 prior to stretch, and then after stretch
00:14:31.12 they align perpendicular to the stretch vector,
00:14:33.21 and you can see this really quite dramatic reinforcement
00:14:37.23 or thickening of the actin stress fibers.
00:14:39.12 Over here in contrast, if we look at the zyxin null cells
00:14:43.21 you can see starting out that the cells are not aligned in any particular
00:14:48.18 way, and what you see after stretch is that they actually are aligned,
00:14:54.21 but you can see quite clearly that they have not
00:14:57.22 generated this really robust actin reinforcement response that
00:15:03.12 characterizes the response of the wildtype cells.
00:15:07.06 And we can see this quantitatively first down here
00:15:09.29 if we look at the alignment index you can see that there is no difference
00:15:14.01 really between the behavior of the wildtype cells and the zyxin null cells.
00:15:17.27 However, over here if we look at the actin
00:15:21.29 thickness index, you can see that the wildtype cells
00:15:27.08 in the closed circles really nicely reinforce their actin
00:15:32.13 cytoskeleton in response to stretch.
00:15:34.18 Whereas the zyxin null cells although they are re-aligning,
00:15:38.03 really don't dramatically increase the thickness of their actin filaments
00:15:41.18 in response to stretch.
00:15:44.29 So this told us really two very important things. First of all,
00:15:48.16 it told us that zyxin was required for the cytoskeletal reinforcement
00:15:56.12 that occurs in response to mechanical force,
00:15:59.04 and this was I think a very important new insight into
00:16:02.14 how cells respond to mechanical stimulation.
00:16:06.26 It also told us that zyxin was not responsible for cytoskeletal re-orientation,
00:16:13.16 and that then allowed us to distinguish between these two
00:16:18.19 possible models here and clearly illustrates
00:16:21.15 that re-orientation does not depend on reinforcement
00:16:25.11 and suggests that these two
00:16:29.09 responses are mechanistically distinct.
00:16:32.23 So thinking about...where is the sensor?
00:16:40.15 Obviously zyxin is at focal adhesions, so it seems reasonable to think
00:16:45.28 that the initial sensory device is housed within the focal adhesion
00:16:52.02 and one candidate for the molecular sensor,
00:16:56.13 or at least a molecule that really contributes
00:16:59.10 to the ability to sense these changes in mechanical stimulation
00:17:03.00 is the integrin adhesion receptor,
00:17:07.19 because clearly that is the transmembrane link between the
00:17:11.18 extracellular matrix and the actin cytoskeleton.
00:17:14.02 So it's positioned in sort of almost the perfect location
00:17:19.11 to be the conduit for sensing these mechanical changes.
00:17:23.10 So we looked to see whether or not integrin dependent
00:17:27.06 adhesion was required for the ability of zyxin
00:17:30.18 to move from the focal adhesions to
00:17:33.08 the actin cytoskeleton in response to stretch.
00:17:37.12 And the way we manipulated the adhesion of the cells
00:17:40.19 is to plate cells on polylysine, which allows the cells to spread, but they
00:17:45.21 do not require integrin dependent adhesion.
00:17:48.20 And here you can see that in unstretched cells plated on polylysine
00:17:53.06 you see zyxin in these concentrated adhesive structures,
00:17:57.29 and when we stretch these cells,
00:18:01.15 in contrast with cells plated on a fibronectin or collagen matrix
00:18:06.11 that engages the integrins,
00:18:08.12 where zyxin would move to the actin cytoskeleton, what we see is that the zyxin
00:18:11.28 protein really does not change its distribution in response to stretch.
00:18:17.27 So this experiment illustrated that zyxin depends on integrin
00:18:24.03 engagement in signaling in order to
00:18:25.29 sense the cues that are coming when
00:18:30.02 cells are exposed to mechanical stimulation.
00:18:33.03 So we see now that cells respond to mechanical stress by enhancing
00:18:40.03 their cytoskeletons, reinforcing their cytoskeletons
00:18:43.18 in a very dramatic way, and in a way that depends on integrin dependent adhesion
00:18:48.19 and the focal adhesion protein zyxin.
00:18:52.13 And we know that cytoskeletal reinforcement is actually really
00:18:59.21 regulated in general by the small GTPase Rho, which is
00:19:06.05 required for the ability of cells to build stress fibers. And so we were interested
00:19:10.18 in whether or not there was a role for Rho in this cytoskeletal
00:19:15.23 reinforcement process, or in the reorientation process,
00:19:20.11 but basically, what is the role of Rho in the response to mechanical stress?
00:19:24.09 And we tested this by exploring the impact of
00:19:28.11 inhibiting one of the downstream effectors of Rho, the Rho-kinase,
00:19:31.13 and analyzing how cells responded to mechanical stimulation
00:19:35.22 when Rho-kinase was inhibited.
00:19:37.25 And here you can see again on the top panel control cells
00:19:41.23 either exposed to...unstretched or exposed to stretch,
00:19:46.10 and once again, the very dramatic
00:19:48.22 reinforcement of the actin cytoskeleton
00:19:51.06 and realignment of the cells perpendicular
00:19:54.24 to the stretch vector. And the surprising thing
00:19:57.26 that we observed when we inhibited the Rho signaling pathway, in particular
00:20:01.26 Rho-kinase, is that not unexpectedly, the unstretched cells
00:20:08.16 didn't have very robust actin cytoskeletons,
00:20:11.06 but surprisingly, when we stretched those cells they actually
00:20:15.18 responded many of them, by building these actin filament
00:20:21.14 bundles that interestingly were not oriented perpendicular to the stretch vector.
00:20:27.16 So these results I think were both striking and surprising.
00:20:32.23 And suggests that actually the Rho signaling pathway may be important for
00:20:38.09 the cell's ability to reorient the cytoskeleton in response to stretch
00:20:45.17 but does not appear to be critical
00:20:48.03 for the cytoskeletal reinforcement.
00:20:51.19 So what are the mechanisms by which the cytoskeletal reinforcement
00:20:57.04 might occur? We see this thickening of the actin stress fibers. This could occur
00:21:01.17 by recruitment of filamentous actin from the
00:21:05.29 cytosol into the actin stress fibers.
00:21:08.16 It could occur by increasing actin bundling along the
00:21:12.17 length of the stress fibers, stabilizing preexisting
00:21:16.29 actin filaments against depolymerization
00:21:20.08 or perhaps stimulation of de novo actin assembly
00:21:24.27 and polymerization. And we wanted to explore this possibility
00:21:30.16 and assess which other factors might collaborate with zyxin
00:21:35.06 in this cytoskeletal reinforcement process.
00:21:38.02 And you may recall from part two of my presentation that I talked with you
00:21:44.24 about the fact that the zyxin protein has a structural and functional similarity
00:21:51.06 to the Listeria ActA protein which is critical for enabling actin
00:21:57.29 filament assembly on the surface of this intracellular
00:22:00.19 pathogen, Listeria. And zyxin in particular has
00:22:06.04 four of these ActA repeats which are proline rich
00:22:09.04 repeats which are responsible for docking Ena/VASP proteins which are
00:22:15.13 clearly implicated in the regulation of actin assembly and
00:22:18.29 which contribute to enhancement of actin assembly.
00:22:22.05 So as an initial step to explore whether or not Ena/VASP proteins
00:22:26.28 might be involved as well as zyxin
00:22:29.18 in this cytoskeletal reinforcement in response to mechanical stimulation,
00:22:34.18 we explored whether or not Ena/VASP proteins like
00:22:40.01 zyxin move from the focal adhesions to the actin cytoskeleton
00:22:44.12 in response to stretch. So we first wanted to explore whether
00:22:49.13 or not the VASP protein responds to mechanical stress
00:22:53.16 by mobilizing from focal adhesions
00:22:56.22 to the actin stress fibers. And we did this by looking at wildtype cells,
00:23:04.08 and here you can see that VASP is normally localized
00:23:07.16 in focal adhesions of unstretched cells
00:23:10.10 where it's co-localized with the zyxin protein.
00:23:13.11 And then when you stretch those cells,
00:23:16.15 quite strikingly, and very similar to what we observed for zyxin,
00:23:20.00 the VASP protein moves to these actin filament arrays.
00:23:25.19 So like zyxin, VASP is mobilizing from focal adhesions to the actin stress fibers
00:23:30.22 very dramatically in response to this uniaxial cyclic stretch.
00:23:37.00 So since we know that zyxin interacts directly with VASP, we looked to see
00:23:43.09 whether or not this VASP redistribution
00:23:46.12 in response to stretch depends on the zyxin protein.
00:23:50.04 And so here we have examined the behavior of VASP
00:23:55.05 in zyxin null cells that have been
00:23:57.07 stretched. And what you can see is that already in the
00:24:01.19 unstretched cells VASP is absent from the focal adhesions
00:24:06.18 illustrating that VASP depends on zyxin to accumulate at these
00:24:11.21 focal adhesion sites. And likewise, after stretch, we don't see any accumulation
00:24:17.12 of VASP on the actin stress fibers in the zyxin null cells
00:24:22.03 compared to wildtype cells,
00:24:23.18 consistent with the idea that VASP really depends on zyxin
00:24:29.09 for both of these subcellular localizations.
00:24:32.13 Both localization to the focal adhesions in unstretched cells
00:24:36.02 and localization to the actin stress fibers in stretched cells.
00:24:41.04 And we can demonstrate this directly by re-introducing into the zyxin null cells
00:24:46.29 a GFP tagged zyxin, and under those conditions, we can see that VASP
00:24:54.09 now shows restored localization in the focal adhesions
00:24:59.05 and to the stress fibers upon stretch.
00:25:02.04 So clearly VASP is responding coordinately with zyxin
00:25:08.29 in response to stretch, and actually depends on the zyxin protein for its ability
00:25:16.05 to mobilize from focal adhesions to actin stress fibers in response to stretch.
00:25:22.13 A really interesting question that is not resolved
00:25:25.06 is whether or not the Ena/VASP proteins are absolutely essential
00:25:29.28 for this cytoskeletal reinforcement response.
00:25:33.01 And so we are hoping to approach that in two ways,
00:25:36.04 first to examine the response of Ena/VASP null cells
00:25:42.04 to uniaxial mechanical stretch and to see
00:25:46.08 whether or not these cells that lack Ena/VASP family members
00:25:50.10 will reinforce their actin cytoskeletons as wildtype cells do.
00:25:54.17 And secondly, since we have been able to show that we can reintroduce
00:25:58.03 a wildtype zyxin protein into zyxin null cells and reconstitute
00:26:04.08 a normal stretch response, we are developing mutated forms of zyxin
00:26:10.14 that lack functional ActA repeats.
00:26:13.15 And we will be able to reconstitute our zyxin null cells
00:26:16.11 with a zyxin molecule that is functional except for
00:26:20.21 its ability to interact with Ena/VASP family members
00:26:24.02 and explore in those cells whether or not the cells respond
00:26:29.03 appropriately to uniaxial cyclic stretch.
00:26:32.10 So in conclusion then what we have been able to show is that the zyxin protein
00:26:40.16 is really, really important for the cells' ability to respond to mechanical stimulation
00:26:49.00 and in particular, to reinforce
00:26:52.09 the actin cytoskeleton in response to a mechanical stress.
00:26:58.16 This mechanical stress activates these two very important responses:
00:27:03.29 the cytoskeletal reinforcement response and a reorientation response.
00:27:09.01 The integrin proteins and zyxin
00:27:12.11 are important in this cytoskeletal reinforcement pathway and likely
00:27:18.17 the Ena/VASP family members will be as well
00:27:21.10 because they facilitate zyxin's ability to stimulate and promote actin assembly.
00:27:27.25 And we've shown that they move with zyxin in a coordinated fashion
00:27:32.08 in response to mechanical force. Interestingly,
00:27:36.19 this second response to mechanical force, the cytoskeletal re-orientation
00:27:43.21 we were able to show is independent of zyxin
00:27:47.04 but rather seems to be dependent on the activity of Rho kinase.
00:27:52.28 And we believe that one of the things that is happening
00:27:56.16 is that zyxin and VASP are recruited to the actin cytoskeleton in response to
00:28:03.13 stretch and that allows, because of their properties
00:28:07.27 in inducing actin assembly, that allows for reinforcement
00:28:12.23 of these actin stress fibers.
00:28:14.28 There are many, many more questions that remain to be addressed here
00:28:20.11 in this extremely interesting
00:28:23.01 research area. Again, I think that this
00:28:26.23 whole area of how cells respond to mechanical cues
00:28:30.07 is really a relatively new frontier in
00:28:32.19 cell biology that has important biomedical implications
00:28:36.25 since the inappropriate cellular and tissue response to stretch
00:28:46.18 or mechanical stress can lead to a variety of different types of
00:28:50.05 pathological situations. And we hope that
00:28:55.18 we are going to be able to understand more about exactly what
00:28:58.27 is happening in these situations by understanding what are the signals
00:29:03.03 that emanate from these integrin rich adhesions sites
00:29:07.16 to zyxin and its partners to stimulate their redistribution and
00:29:11.19 perhaps their change in activity, which leads to this important
00:29:15.27 cytoskeletal reinforcement response.
00:29:18.28 So again, we've got zyxin and its partners playing critical roles
00:29:24.19 in the cellular response to mechanical stress,
00:29:27.15 and I think these results really focus our attention on these
00:29:32.21 adhesion plaques, or focal adhesions,
00:29:35.06 as domains that are important domains in the ability to sense
00:29:40.21 mechanical cues, and really this leads me back
00:29:45.20 to one of my introductory comments
00:29:47.15 about homeostasis, and you begin to think about the ability
00:29:52.15 of cells to have tension homeostasis and maintain
00:29:56.29 a balance of force at these adhesion sites. Under normal
00:30:01.18 conditions you would imagine that there is a balance achieved with
00:30:05.27 the extracellular matrix and the actin stress fibers,
00:30:09.12 and if you actually pull on the extracellular matrix
00:30:16.17 or build up a lot more extracellular matrix which impinges on the cell
00:30:20.16 then the cell responds to that perhaps in a way that is similar to
00:30:24.10 what occurs with uniaxial cyclic stretch
00:30:26.29 by reinforcing the actin cytoskeleton.
00:30:30.06 And I think this is telling us that cells are really
00:30:33.18 able to sense this delicate balance
00:30:36.04 here and achieve a balance of forces
00:30:39.25 at these important sensory zones for the cell
00:30:44.10 So thank you very much, and
00:30:46.18 I look forward to being able to provide you an update on this
00:30:50.14 interesting research project in the future.

Videos in this Talk

Part 1: Adhesion, Signaling and Cancer
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 2: Discovery and Characterization of a Focal Adhesion Protein Implicated in Tumor Progression
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 3: Focal Adhesions as Stress Sensors
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Mary Beckerle

Total Duration: 1:57:53

Recorded: July 2007

Educator Resources for this Talk

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Talk Overview

Cell-substratum adhesion is mediated by integrins, a family of transmembrane, heterodimeric, extracellular matrix receptors that are concentrated at focal adhesions. Integin engagement influences a variety of signaling pathways and regulates cell behaviors including motility, proliferation, and survival. Disturbance of normal integrin function is associated with a variety of pathologic conditions including cancer. In the first segment of my seminar, I provide a broad overview of the cancer problem for a lay audience. Advances in our understanding of cancer as a genetic disease are discussed. The influence of cell adhesion on control of cell growth is reviewed.

In the second segment, I describe the identification of the focal adhesion protein, zyxin, by my lab. Recent work revealed that zyxin is down-regulated upon expression of the Ewing sarcoma oncoprotein, EWS-FLI. Loss of zyxin expression results in enhanced cell motility and is also associated with failed apoptotic signaling. Evidence that zyxin shuttles between focal adhesions and the nucleus is presented. The impact of reduced zyxin expression on tumor progression is discussed.

In the third segment of my seminar, I address a new frontier in cell biology, that is how cells respond to mechanical information. Cells and tissues are exposed to physical forces in vivo and excessive mechanical stress leads to a variety of pathological consequences. I describe a system for exposing cells to controlled mechanical stress and discuss the stretch response. We have discovered that the focal adhesion protein, zyxin, is exquisitely sensitive to mechanical stimulation and is required for the ability of cells to reinforce the actin cytoskeleton when challenged by exposure to cyclic stretch.

Speaker Bio

Mary Beckerle

Mary Beckerle is Professor of Biology and Oncological Sciences at the University of Utah. Dr. Beckerle serves as the Executive Director of Huntsman Cancer Institute and holds the Ralph E. and Willia T. Main Presidential Endowed Chair. She obtained her undergraduate training at Wells College and earned a PhD in Molecular, Cellular, and Developmental Biology… Continue Reading

Part 1: Adhesion, Signaling and Cancer

Part 2: Discovery and Characterization of a Focal Adhesion Protein Implicated in Tumor Progression

Part 3: Focal Adhesions as Stress Sensors

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Mary Beckerle

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