 Good afternoon, folks. If everybody would take their seats, I think we'll go ahead and get started. So welcome. Good to see all of you. I'm Eric Green, Director of the National Humidgenome Research Institute, and I want to welcome you to this year's annual Trent lecture. I'm going to just make a few introductory remarks about this lecture, and then I'm going to turn the podium over to NHGRI's scientific director, Dan Casper, who will introduce this year's speaker. By way of background, for those of you who don't know this, the Intramural Research Program of NHGRI was started in 1993. It started when Francis Collins was recruited to come here to be the director of the institute, and at that time, part of the deal for him to come here was his ability to create, establish, from essentially the ground level up, a new intramural program that would be part of the institute. For that, he recruited his friend and University of Michigan colleague, Jeff Trent, to join him in moving to Bethesda and to be the very first scientific director for NHGRI, otherwise known as the director of the Division of Intramural Research, and to some extent the rest is history. So Jeff arrived here in 1993 with essentially nothing in terms of an infrastructure for having an intramural program, and built one up very quickly, looking around, involving a number of you who came here as part of the first wave of, at that time, young investigators. You folks don't look nearly as young as you did in 1993. I arrived a year later, but I was essentially recruited about the same time, just delayed my move for a year, and was able to watch firsthand Jeff's ability to build a vigorous research program very quickly from the ground up. Part of that was a combination of being really wise and who he and Francis recruited to be the first set of investigators, and part of it was his incredibly good organization, management skills, good scientific taste, and so forth. Jeff served as the scientific director for NHGRI from 1993 to 2002, at which time he departed NIH to be the founding director of the Institute of Genome Research out in Arizona, and where he has continued to use his incredible skills to build research programs and be incredibly productive. Shortly after his departure, I was appointed the second scientific director of NHGRI, and one of the very early things that I did was to find some way to thank Jeff in perpetuity for his founding directorship of the intramural program, and I thought what he would like, and indeed I think he's told me he has liked the idea of making it scientific, and so we created an annual lectureship in his name. In fact, it's the only annual lectureship we have at NHGRI to this day, and it basically honors his contributions as the first scientific director. So each year we look to invite a star in the field of cancer, especially where cancer meets genomics, certainly this was and continues to be Jeff's passion, and it seems that every year we're successful at identifying such a first-rate scientist to come here, tell us about their research, and really showcase the exciting opportunities there are in cancer research, and especially as it sort of continues to grow and thrive with genomic advances. So that's the history, and I'm going to now turn the podium over to Dan Kasner, who's going to introduce this year's Trent Lecturer. Thank you. Well, thanks very much, Eric, and it's my tremendous honor and pleasure to have the opportunity today to introduce this year's Jeffrey M. Trent Lecturer, Dr. Joan Brugge. Dr. Brugge is the director of the Ludwig Cancer Center at Harvard University, and is also a professor of cell biology at Harvard. Joan did her undergraduate work at Northwestern University, and then went to the Hall of Halls of Baylor College of Medicine and worked with Janet Butel for her graduate work, then going on to do her postdoctoral work at the University of Colorado with Dr. Raymond Erickson. She started her faculty career at Stony Brook, SUNY Stony Brook University, going on to the University of Pennsylvania as a Hughes investigator, becoming the chief scientific officer of Ariad Pharmaceuticals before joining the faculty at Harvard in 1997, I believe. And at Harvard, she was in the Department of Cell Biology, and has been ever since, was the chair of the Department of Cell Biology for 10 years before taking her current position as the director of the Ludwig Institute in 2014. She started her work on cancer in earnest as a postdoc studying the cellular homologue of the SARC gene, looking at it in terms of its role in signaling, and looking at the HSP proteins as chaperones for the cellular SARC gene, went on then to look at integrin-induced signaling and platelets in the role of tyrosine kinases in that process, and then on to a very, very storied and productive career looking at signaling in cancer. She is the recipient of numerous awards and honors, including the NIH Merit Award, election to the American Academy of Arts and Sciences, election to the National Academy of Sciences and Medicine. So today, she is going to be regaling us on another topic of great interest, and that is looking at the TRPA1 gene and the calcium-sensitive channel encoded by it in oxidative stress responses to cancer. So Joan, thank you very much for coming, and we look forward to your talk. Thank you, Dan. It's really a pleasure to be here. In fact, it's an honor to be the Trent Lecturer. I think it's a real tribute to or testament to Jeff Trent's both good taste as well as his vision that the NHGRI has been so successful, and it's really been fun for me not only to see old friends, but also to hear about science that I'm really not familiar with and just fascinated by in the discussions I've had this morning. So today I'm going to tell you about a story that started a while ago when I first came back to academics after having been and helped us get a biotech company off the ground for a short period of time, and this project was initiated, this project kind of was motivated us, or we were motivated to use three-dimensional cultures in order to understand events that are associated with epithelial cancers. And I'll show you some early studies that kind of perked our interest in oxidative stress and then tell you, spend most of my time on a whole new story unpublished that relates to a protein that we never thought we would ever have any interest in whatsoever, but that actually has shed a lot of new insights into mechanisms of oxidative defense as well as the role of oxidative defense in cancer progression. So this is a more general talk than just on trip A1, more on oxidative stresses of defenses in cancer. Okay, so this story started, as I mentioned, when we first started working in using three-dimensional structures to mimic the organization of cells in the normal breast in order to understand early events associated with tumor genesis. And one thing that we had noticed when we were just using these three-dimensional cultures is that when there were aberrant cells that proliferated, even after these asynyl-like structures had undergone growth arrest, that if they proliferated into the center of the structure, they would die. And so we were being interested in early events associated with carcinogenesis. We were interested in how, in fact, what would be the events that would be associated with the conversion of these kind of hyper-proliferative structures into structures in which the cells would survive in the liminal space since we saw that these aberrant proliferating cells would undergo cell death. So one of the major thrusts of our early studies was, first of all, to understand what causes the death of these cells. And then secondly, what types of oncogenic insults would allow these cells to survive in the center of the structure to form these carcinoma in situ-like structures, which looked very similar to the carcinoma in situs that are seen in one type of epithelial tumor, glangular tumors. And so I'm just going to summarize what we learned. And a lot of this was motivated by Jay Devneth, who actually got his research start here at NIH when he was a medical student. He did one of the two-year HHMI internships in Harold Varmus' lab. He actually worked with Pam Schwarzenberg. So Jay ended up going on to a pathology residency at the Brigham, and then he did his research in our lab. And I think it was really Jay's kind of keen pathological sense of attention to visual cues that really led him to provide some really interesting insights in the mechanisms of morphogenesis of these acenylyte structures. So Mina Bissell's lab and Panda Klinman's lab also here, she's really the hero of these three-dimensional structures. And she worked out that if you put a salivary epithelial cells into extracellular matrix gels, you could generate these acenylyte structures. But what Jay found is that he followed the mechanisms associated with the morphogenesis, which initially started as just the formation of a solid ball of cells and then underwent spatially and temporally regulated events that led to the formation of growth-arrested polarized hollow structures. And a key insight that Jay made was that the formation of a lumen actually involves selective apoptosis of the cells in the center of the structure. You can see here that these cells, this is in early stages, when here you see, you know, when there's a solid sphere, and then the cells in the center undergo apoptosis. And one thing that he noted in staining with a lot of different markers is that signals that are downstream from epidermal growth factor, which is the growth factor that's used in these cultures, after the cells underwent a polarization, the outer cells polarized and the inner cells didn't. The signals to activate signaling through the PI3 kinase and ERT pathway were specifically localized only in the outer cells. And we prepostulated that these inner cells that were actually deprived of integrant signals from the, after the extracellular matrix protein deposition became polarized such that only the outer cells received the signals from the integrant that these cells were basically starved of survival signals and underwent cell death. So one of the first questions that Jay asked was, what would happen if we would prevent apoptosis? Would that maintain those cells in the center of the structure? So Jay just overexpressed BCL2 and BCLXL in order to block apoptosis and then followed the progression of morphogenesis. And what he found was that for the first week after expression of BCL2, BCL, or BCLXL, that there was no apoptosis and that the inner cells were intact. But then he happened to go back to the cultures and look at them a few days later and found that they were completely hollow even though there was no evidence that there was a breach in which allowed apoptosis. So we wondered what was responsible for this later stage clearing of the luminal cells. And one thing that Jay noticed is by doing transmissional electron microscopy that the cells in the center of the structure, and these are the outer cells, these are the inner cells, were just loaded with autophagic vesicles or autophagyl lysosomes. So autophagy is a process activated to a great extent when cells are starving. So he postulated that these cells may be starving and that's one reason why they undergo this process. And presumably this would lead eventually if the salvation wasn't resolved to a necrosis like death. So he proposed that the cells would then undergo a necrosis like death. So after Jay left, I tried for a long time to find someone who was willing to trace the metabolic fate of these cells in the center to see if in fact they were starving and if there were metabolic defects associated with the cells in the center. And it actually took five years before I found someone who was willing to do that and that was Zach Schaefer who was a student, graduate student at Duke in his lab. They started working on some aspects of metabolism and so he was willing to take on the challenge and then a graduate student, Alex Grosjean, joined him. And to make a long story short, what he found or so one thing is difficult to follow metabolism in a three-dimensional structure. And since all of the lines of evidence we had was that the cells in the center were undergoing these, eventually underwent apoptosis because they were not receiving integrant signals from the outside. He started by just looking at metabolic defects associated with survival of cells in suspension without attachment to matrix. And using a whole variety of different experiments, he found that in fact that when cells are not attached to matrix they're unable to transport or they, there's a dramatic decrease in both glucose and amino acid uptake. And this then when he followed it by flux studies led to a significant decrease in glycolysis and TCA cycle as well as pentose phosphate cycle, flux through the pentose phosphate pathway, significant reduction in ATP and an induction of reactive oxygen species. So one question was would there be a similar, would there be similar changes in the three-dimensional structures? And we really couldn't look at ATP at that time. Now there are some imaging approaches that make that somewhat feasible. What we did was to work with Lohling Song who is a two-photon microscopist and just looked at this really early stage before there was any sign of any cell death, looked by two-photon microscopy which allowed us to look at just the overall levels of NADH and NADPH. And you could see that there was a dichotomy between the inner and the outer cells. We didn't know what caused it but we could see that there was, there were differences in metabolic activity of these. But then when we used a stain that fluoresced in the presence of reactive oxygen species, DCFDA, you could see that the inner cells were loaded with reactive oxygen. So this suggested that perhaps the same kind of events were going on. And then to address whether the reactive oxygen contributed to the elimination of the cells, Jay stained, I mean Zach treated the cells with several different antioxidants and sure enough treatment with NSTL cysteine or Trolox blocked the death of those cells in the center of the structure, suggesting that Ross was actually responsible for the elimination of those cells. And then he actually went on to show that in fact what was known at the time from Craig Thompson's lab is that if cells were attached to matrix and you starved them intentionally of glucose and amino acids, they would survive because they would up-regulate fatty acid oxidation. So they'd use fatty acids as a substrate instead of amino acids and glucose. And what we found was that in suspension the cells were unable to up-regulate fatty acid oxidation. So basically they were starved for glucose, amino acids and they couldn't use fatty acid. And in fact under conditions which we treated with antioxidants that rescued fatty acid oxidation and the cell survived. So it all suggested that it was actually Ross was specifically allowing the elimination of cells that were starved of glucose and amino acid when they're outside of their natural environment. So we think this is kind of a homeostatic process that's part of the nature's way of eliminating cells that are outside of their natural matrix, I mean outside of their natural environment. Now apoptosis is the most rapid way to do that. But then when apoptosis is faulty, ROS is like a backup mechanism to get rid of those aberrant cells. We were interested in whether this was anything to do with natural events. And so we looked at a process that we believe might mimic the process that we saw in the three-dimensional structures which is the formation of a lumen in the pubertal mammary gland. So in the pubertal mammary gland, you have these branches that elongate to form the ductal system, the virgin ductal system. And the way in which these branches form is that there's proliferation at the tip. These tips are called terminal end buds. So you're continually adding cells here which basically creates a solid mass. In order to have the duct, you have to eliminate these cells. And Jeff Rosen's lab showed many years ago that he proposed that the lumen was formed by apoptosis because he saw evidence of apoptosis just on the inside of these terminal end buds. And so we had found in the, in our three-dimensional structures that a pro-apoptotic protein called BIM was necessary for clearing of the lumenal space by apoptosis. And so we got BIM knockout mice from Andreas Strasser and then looked at the clearance of the lumen in the BIM knockout mice. Okay. And this was done by Androma and Arnaud Mellieu who was a post-doc in the lab. And so basically this was in a BIM wild type. You have this clearance in apoptosis. And I'd say it mentioned if you overexpress BCL2 or if you knock down BIM, you prevent apoptosis. And the results were really striking. We actually didn't expect this because there's other BH3 only proteins that could potentially be involved. But basically 93% of the terminal end buds were solid clubs. There was no clearing of the lumenal space at all. But what we found was interesting is that eventually there was clearance of the lumenal space. And if you look by H&E, what we saw was that just at the point where the outer cells, the lumenal cells, polarized and basically they lose their adhesive surface, that that isolated these cells that were in the center. And they eventually just underwent what looked like a necrotic type death. And if you stained with an antibody against 4 H&E, which is a lipid peroxide product that is generated under conditions of high reactive oxygen, you see that there is very high levels of lipid proxidation in these cells in the center. So we proposed that they were undergoing a Ross-mediated cell death, so very similar to what we had seen in the BCL to expressing cells over time. So we think that this process of elimination by necrosis due to likely starvation, so after these cells are kind of isolated, when the outer cells polarized, they're likely deprived of nutrients, probably growth factors as well. And this leads to this necrotic-like death. So this is likely, you know, why a number of sculpting events still occur under conditions in mice where you block apoptosis because this other mechanism takes over. So again, it suggests that it's an alternate backup to ensure natural morphogenesis under conditions in which the most physiologic means of eliminating those cells is disrupted. Okay, so basically that suggested, what that suggested to us was that in order for tumor, or initiating early tumor cells to be able to fill the luminal space, that they would have to have an anti-apoptotic insult to prevent apoptosis, but then they would need another event that would rescue the metabolic impairment of those cells that were, which did not die by apoptosis. So we looked at a lot of different oncogenes and to see which oncogenes were capable of conferring these different activities. And like had been shown by many people, the ERC, PI3 kinase, NFKappa B pathway when super activated, say by constitutive activation, or mutants that lead to constitutive activation, we could prevent apoptosis. But interesting, of the ones that we looked at, only those oncogenes that activated the PI3 kinase, AKT pathway were able to rescue the metabolic impairment. And what we found was that the reason that they were able to rescue the cells, the metabolic impairment is, as I mentioned before, the cells when they're detached from matrix, they can't transport glucose or glutamine. However, when there's an activated PI3 kinase pathway, this leads to constitutive activation of AKT, which is required for glucose and glutamine transport. So if you prevent, when the cells are detached, you don't get activation of PI3 kinase because you need integrant signals to co-activate the PI3 kinase pathway with the EGF receptor activation. But when you activate AKT, you rescue the need for integrants. And so the cells can transport glucose and glutamine. So they're rescued. And then what we found as well is that any oncogenes that led to constitutive activation of the PI3 kinase, AKT pathway, would also rescue the metabolic impairment. So we think this is one reason why the PI3 kinase pathway is so commonly activated in cancer, because it not only is a very strong, not only confers very strong anti-epoptotic activity, but also can rescue this metabolic impairment. OK, so what are the implications of this for early development? So one we would propose is that when there's a hyper-proliferative insult that leads to hyper-proliferation of the cells, this would, the cells would proliferate in the center of the structure. And those cells that proliferate in the center of the structure would undergo apoptosis. If there was an insult that prevent that an anti-epoptotic checkpoint, an apoptotic checkpoint was lost, the cells would survive. But we would propose that they would be metabolic impaired due to lack of nutrients and they would undergo cell death. And so the abnormal cells would die. So you have both of these these checkpoints intact to prevent cancer from developing. Now what we would then surmise is that both is that oncogenes like or B2 and PI3 kinase prevent apoptosis and they rescue the metabolic defects. So they would theoretically allow survival. But we also I also mentioned that both of these oncogenes are known to increase the levels of cellular ROS because of effects on cellular metabolism. So there's also ROS that's induced to some extent by oncogenes as well. And I'm just mentioning this because it appears so one would propose then that in this context where you don't have a strong oncogene that's rescuing the metabolic impairment that up regulation of antioxidants could prevent the ROS killing. But even in the context in which you have activation of oncogenes there's evidence suggesting that there still may be a selection for cells that have upregulated antioxidant gene expression programs. So ROS wouldn't be generated because of a lack of the nutrients, but it would be generated by other metabolic consequences of having these activated oncogene pathways. So in either case there's selective pressure potentially or opposed for up regulation of antioxidants then lead to tumor progression. So we published this paper around 2009 and we got a lot of kind of negative feedback because this seemed to challenge the dogma at the time that antioxidants are tumor suppressants. And this was because ROS is also known to induce DNA damage and mutations. So ROS can also contribute to initiation of tumors by inducing DNA damage and then leading to tumor initiation. And so what we're proposing that is if you and so if you block ROS with antioxidants this could prevent tumor initiation. But what we're proposing is that after tumors are initiated, antioxidants would not be suppressive but actually be promoting for tumor progression. And since this time there's lots of evidence now that indicates that following tumor initiation that antioxidants can be strong promoters of tumor progression. I'm just going to mention a few things here. So one of there have been multiple multiple epidemiologic studies similar to the one that I show here. So this was a trial and then enrolled 35,000 men in which they were treated with vitamin E or placebo. And then they looked at the induction of prostate cancer. And what you can see is you didn't see much at first but then they separated very significantly and those patients or those men on the study that had vitamin E had a higher incidence of cancer. And there have been several other large epidemiologic studies that support this as well. So this epidemiologic evidence that antioxidants can be pro-tumorogenic. And then there have been multiple mouse models. I'm just going to show you two here. This was a very direct assessment of dietary supplementation with antioxidants. So this is a study from the Bergo lab where they took a BRAF lung tumor model, BRAF mutant lung tumor model. So this is the normal incidence of cancer and this is the incidence of cancer when the mice were fed either anesthetial cysteine or vitamin E. So you can see there's a very significant acceleration of tumor progression. And then this is a study from Dave Tuvason's lab where he did the opposite. So their lab had found that KRAS activated NRF2 which is a transcription factor that's kind of known to be the major, regulate the major antioxidant transcriptional program. So it activates multiple antioxidant programs as well as other xenobiotic detoxification programs. So they found in their pancreatic model, KRAS activated NRF2. So they wondered whether NRF2 was contributing to tumor progression. So they crossed their KRAS mutant mouse with either NRF2 knockout mice and found that it should be minus, minus. There was a dramatic inhibition of tumor progression. So when they blocked the antioxidant program, they blocked tumor progression which would support the idea antioxidants are pro-tumorgenic. And then the best evidence for a really critical role of antioxidants in some tumors is the evidence that these three genes that regulate the stability of NRF2 and they're the major regulators of NRF2 stability and nuclear localization that there are mutations in NRF2 keep and call three that lead to stabilization of NRF2 and activation of this whole program. And multiple tumor types show a fairly significant level of mutation and these are generally tumors that are in the airway like the long both adenone squamous and then other squamous tumors that are exposed to air along the airway. So this is really the best evidence that up-regulation of antioxidants can be tumor promoting. So there's lots of people studying these mycine defective in the, or hyper-activated NRF2 to identify ways of blocking it. Okay. But there's evidence suggesting that NRF2, mutations in the NRF2 axis aren't the only way of regulating antioxidant programs. I'll just show you here. There are no mutations in NRF2, call three or keep in breast tumors but you can see here this is just, Isaac Harris is a postdoc in the lab made a list of 150 different positive or negative regulators of oxidative stress and you can see that the basal triple negative breast tumors have high levels of antioxidants and low levels of the pro oxidants. Her tube tumors are also high so there are other mechanisms that lead to activation of antioxidant programs in addition to NRF2. Okay. So then just overall, that's kind of the end of my introduction, there are multiple different aspects or features of the process of tumor genesis that involve the generation of ROS. So tumors up regulate antioxidant programs to prevent unopposed ROS and cell death. Okay. All right. So now I'm going to talk about the unpublished work that was in the title. So what I'm going to tell you about is a completely, you know, unexpected player that plays a role in regulating oxidative stress. And this involves a triple A1 calcium channels. And this work was totally initiated and driven by Japanese postdoc, Nobu Takahashi. And Nobu is a neurobiologist who worked on triple A1 channels both as a postdoc, I mean, as a graduate student and later as a first postdoc in the Mori lab in Japan. So why was he interested in looking at triple A1? Why did he come to our lab? So triple A1 is a member of the triple channel family. These proteins are expressed in sensory neurons and they're responsive to a whole variety of different stimuli that lead to activation of the channel and calcium influx. So the triple channels are particularly interesting with respect to oxidative stress because they have four cysteines in the cytoplasmic tail which when oxidized or reacted with electrophilic agents lead to opening of the channel. So he was interested in, so this was of interest and with respect to oxidative stress. I'll just mention a few other things. So though you may not know it, you are familiar with the consequences of triple A1 activation. So wasabi and mustard oil, the major component of those that give it the strong flavor, a strong irritant is actually AITC which reacts with the cysteines to activate the channel. It's also involved in neurogenic inflammation and asthma and so it's believed to be responsible for activation or for release of inflammatory molecules at the nerve airway interface that leads to the continual irritation associated with asthma and there's actually companies that are developing inhibitors of triple A1 for treatment of asthma, so it's actually an interesting molecule for many other aspects and then also these channels are actually responsible for the pain that's associated with certain chemotherapies and aromatase inhibitors. So these inhibitors actually react with these cysteines leading to the induction of the pain sensation, so it's a very interesting molecule. Most of you probably came into this seminar, a little leery about whether you'd really be interested in triple A1 but it is an interesting molecule. OK, so Nobu was interested in coming to our lab because triple A1 is actually up-regulated in multiple epithelial tumors and in particular it's up-regulated in lung, I mean in kidney, sorry, in breast tumors, well it's kidney tumors and lung tumors. And if you look at its expression specifically in breast tumors, so these are normal breasts and this is invasive ductal breast carcinomas, this is from the TCGA data and this is separated into the different PAM50 subclasses of breast tumors, you can see that there isn't any specific subclass of breast tumors in which it's specifically up-regulated, so it's up-regulated in many and this is just a larger panel of different trip mutants, so you can see that triple A1 is the most commonly up-regulated, although you can see that one of the other TRIP-M channels is also up-regulated specifically in basal tumors. So Nobu was interested in whether the protein was expressed so he got the antibody from the human protein atlas collection that had been very highly validated by them but then he actually knocked out TRIP-A1 in a breast tumor cell line, made tumors and then used those to validate the antibody and it validated very nicely, you see very low levels in normal breast but then in breast tumors there were a reasonable portion of the tumors that were 3 plus but then also we saw 2 plus staining, so there was a significant up-regulation of the protein as well as the RNA in breast tumors and then he also looked in a tissue microarray of lung squamous and lung adenotumors and again the protein expression correlated with the RNA expression, so it looked like the protein was expressed as well. And then just show you, this is a series of different PDX models that we have in the lab, most of them are from Alana Whelms, this is from Jeff Shapiro and Jean Zhang at Harvard but then when we looked at tumor cell lines there was basically only one tumor cell line that had really high levels of expression and just low levels and other ones and you can go into this during the question period, I don't have a lot of time to tell you about this but what we found is that TRIP A1 is actually a downstream target of NRF2 and it's also activated by a variety of different irritants and stimuli, TNF activates at about 400 fold, it's also activated by just oxidative stress, this shows it's up-regulation by oxygen so we think one reason why we're not seeing it as commonly up-regulated in culture is that it's likely up-regulated in the tumors under conditions where there is oxidative stress or potentially other factors like TNF that could lead to its activation. Okay, I just wanted to mention one other one, here's my, I have two genetic slides, so another tumor that had very high levels were these malignant peripheral nerve sheath tumors or MPNST and there was actually 25 fold amplification in these tumors, other amplification in others, we've looked really closely at the breast, the amplification is pretty minimal but it was more significant in the MPNSTs and so we got, and also the level of RNA expression was significantly higher in the tumors which we did not see in the breast so it suggested that this was more meaningful, there are consequences of this activation. Okay, so actually I guess I left it out there and I'll show you later there are multiple, there's up-regulated in neural sheath tumor cell lines as well and that correlates with the amplification. Okay, so one really important question is so it's overexpressed, the protein's there but is it actually functional, can it, does it function as a calcium channel? Oh, okay, so it just, okay. So what we did was to look at cell lines that overexpressed TRIP-A1 and treated those cell lines with AITC which is that wasabi component that activates the TRIP-A1 channel and then this looked in the different cell lines that have different levels of TRIP-A1 so you can see that this 1569's which has high levels of TRIP-A1 there was significant calcium influx after treatment with AITC and then basically the level of calcium influx correlated with the level of TRIP-A1 expression. Also looked at this in the lung tumor cell lines and here's the MPNST cell lines and again if your eyes can follow the protein levels and the level of calcium influx you see that it directly correlates. So these channels are able to be activated in their functional channels in these tumor cell lines. Okay, so one question is are there consequences of loss of expression of TRIP-A1 in the tumor cell lines? And so one of the things that Nobu did was to grow the cells in soft auger. So this is basically a measure of Anchorage independent proliferation and survival and we actually know that there's loss increase as I showed you earlier in cells that are in suspension and what he found was that there was a very dramatic reduction in the number of colonies formed in soft auger in this breast tumor cell line 1569 after with two different hairpins but nicely we could also treat this control cell line with the TRIP-A1 inhibitors so I mentioned their TRIP-A1 inhibitors. One thing great about this project is there's really good reagents inhibitors and stimulators in the channel that we had to work with. And so you can see that both the knockdown and the TRIP-A1 inhibitor dramatically reduced the survival of these cells in Anchorage independent conditions. And this also just shows that you couldn't say for sure whether that was related to oxidative stress. In this case, Nobu treated the monolayers of cells with increasing concentrations of H2O2 and then measured survival. And here, I'm showing you this lung series here because you can see very, and we have so many of them, you can see the dose response to, sorry, this is in the presence or absence of a TRIP-A1 inhibitor. So in the presence of inhibitor, relatively absence, you see much better survival under oxidative stress. So this just kind of confirmed that the cells were less sensitive to oxidative stress if the TRIP-A1 channel was functional. Okay, but what was really kind of much more convincing to me was when I saw a more kind of natural, the natural context of activation of ROS. Okay, so what Nobu did was to take the 1569 cells, the breastline that has a high level expression, and he expressed a genetically encoded ROS sensor called Hyper2, and then he also monitored calcium influx using Fura2. And basically, if you just look at the control here, what was really interesting is that these cells are surviving in the, if you look at the levels of blue, which it's hard to see because the red and yellow are so strong, but there are cells present and there's a solid sphere of cells. And you can see this gradient of ROS from the outside to the inside. So even the cells are surviving, there's an increased gradient of ROS in those cells. And that corresponds to an increase in the level of calcium. So it looks like TRIP-A1 under condition, it's not like TRIP-A1 is present in the outer cells, but you don't see calcium influx and there's a correlation between the level of ROS and the extent of calcium influx. And this looks real because if you knock down TRIP-A1 or if you inhibit TRIP-A1, you lose that Fura2 signal. So I think this is a really nice imaging visualization of the regulation of calcium influx by the TRIP-A1 channel that kind of correlates with ROS. That's just, Nobu's quantified all this, I'm not showing all the quantification because there's so much data, but he's quantified all of these. Okay, so then important question is what happens to those cells when you knock down TRIP-A1 over time? And so what I showed you there was a day five after knockdown or treatment with the inhibitor. This is a day 10 and this is day 15. And one thing that you can see is that if you stain for Caspase III, a marker of apoptosis in the hairpin treated cells, there was increased apoptosis. But I think what's very convincing to me is when you look at just the DAPI stain itself after 15 days, and what you see is that the outer cells are surviving fine with TRIP-A1 knockdown, but basically every single assinist is empty, it's hollow. So it looks like when you knock down TRIP-A1, those cells that have a higher level of ROS are undergoing cell death. But the outer cells, which had very low levels of ROS and no calcium, they seem to be fine. There is a reduced proliferation of those cells. We haven't really looked into that yet, but you can see the key 67 is down a bit. So this is in the 1569 cells. Oh shoot, I just added this because he's now done it on the, I've passed it in the wrong line. He's now done it on the lung tumor cell lines and the PDX model, and you see similar clearing of the inner cell. So we're seeing it in multiple different cancer types that if the cells have high TRIP-A1, then you see this reduction. We don't see that in cell lines that don't have high TRIP-A1. Okay, so what I've shown you so far, TRIP-A1's expressed at high levels in a subset of breast and lung tumors, PDX models, and that TRIP-A1 is activated by natural conditions associated with ROS generation, and the down regulation of TRIP-A1 in tumor cells suppresses colon inflammation and prevents survival of the inner cells in the spheroids. So we still weren't totally convinced, and so another kind of way of addressing this was to do the opposite experiment, and that was to take normal mammary epithelial cells and over express TRIP-A1, and so would over expression of TRIP-A1 allow survival of those cells that die in the center of the structure. And so first, what Nobu did was he introduced TRIP-A1 into the cells and then just look to see if it would be a functional channel by treating with H2O2, and you can see this is just a single cell recording. You can see that TRIP-A1 was able to be activated in the MCF10A normal epithelial cells when overexpressed, and so this, and this is just the collective data from the, from lots of cells. So you see much more significant, if really high concentrations of H2O2, you start getting some calcium influx, but you see the dramatic difference with TRIP-A1 over expression. Okay, so what happens in the asinine? That was the critical question. So in the vector controls, you can see that there is a similar gradient, and these are in early structures when you still have cells in the center, and as we had seen with the DCFDA, you see an increase in ROS, gradient of ROS. And then if you overexpress TRIP-A1, you see increased calcium influx in the cells in the center. And if you block, if you block with the AP-18, you see a reduction in that. But the critical question was, does the TRIP-A1 overexpression allow survival of those cells in the center? And that's addressed here, so with longer term culture. What you see is that in the cell, if you look at caspase three, the cells that are overexpressing TRIP-A1, I forgot to mention, there's multiple mutants that mutations in TRIP-A1, associated with breast cancer, and Nobu's colleagues in Japan made all those mutants, and only one of them actually had an activated phenotype, and it actually has a really interesting mutation, what predicts that it would have a stronger, a stronger, there would be a stronger electrophil... It would be easier, more readily activated, by electrophilic attack. So anyway, it's better at everything with respect to activating the channel. And you can see that the cells in the center not only survive, but also can proliferate, and basically you don't get this clearing of the luminal space nearly to the extent that you get in the control structures when you overexpress TRIP-A1. Oh, I just wanted to mention that, okay, this is a really important point. When you look at the levels of ROSS, there's no decrease in the level of ROSS in these cells that are surviving, and we've looked at this in many different ways, so TRIP-A1 is not neutralizing ROSS, so ROSS is still present at the highest, at the similar level, but the cells are basically able to survive the insult of the ROS when the TRIP-A1 channel, when you get calcium influx brought about by the TRIP-A1 channel, okay? So no decrease in ROSS, but we get survival. Okay, all right, so this kind of just summarized. So overexpression reduces the sensitivity of MCF-10A cells to H2O2. It prevents the cells from death and suspension. It protects the inner cells in 3D. I didn't show you this, but we see this as well, but TRIP-A1 does not neutralize ROSS. So how's it working? Oh, okay, one other thing I wanted to mention before you show how it works. So as I mentioned before, certain chemotherapies that are strong electrophiles are able to activate, whoops, are able to activate the, able to activate TRIP-A1. So basically, for instance, platinum drugs activate TRIP-A1, and so if you think about if a cell has high expression of TRIP-A1, when you treat with a platinum drug, the high levels of TRIP-A1 will potentially allow the cells to survive the insult of the chemotherapy because it's able to activate these calcium channels and give protection to the oxidative stress. Okay, so one question is does TRIP-A1 protect cells from chemotherapeutic drugs that activate TRIP-A1? So the first question was do we see activation of TRIP-A1 in the breast tumor cells that express high levels of TRIP-A1? So does carboplatin activate TRIP-A1 in these cells? So if you look at the single cell recording, you can see that carboplatin does activate calcium influx and if in the two cell lines that have TRIP-A1 knockdown or TRIP-A1 inhibition, you lose that. So it looks like TRIP-A1 is the only calcium channel that is able to be activated by carboplatin in this particular cell line and that's just a quantification there. So then the question was does this protect the cells from oxidative stress? And the way in which he addressed this was to treat with carboplatin at different depth, just at a dose response curve with carboplatin and looked at the relative viability. In this particular cell line, you can see the shift of the curve. So there is a highly statistically significant, he did this like innumerable times in order to be sure of the result. But if you look in the lung cell lines and in the neural sheath tumor cell lines which have a larger number of and higher expression, you can see that a much more significant shift and it totally correlates with the level of expression of the TRIP-A1. So it looks like TRIP-A1 can protect these cells from carboplatin and presumably because activation, the calcium channel is protecting these cells. And then Nobu addressed this in an experimental model. So he took the HCC1569 cells, the breast tumor cell line injected those cells either that expressed a control hairpin as well as either the two different SHRNAs and he then treated each one with or without carboplatin. So it turns out we didn't realize this but HCC1569s are super resistant to carboplatin. So this is minus and plus carboplatin, basically resistant. If you just knock down TRIP-A1, if you thought those are these curves here, knock down of TRIP-A1 allows the cells to grow better. I mean, they can't grow as well. So TRIP-A1 is likely contributing or the protection against oxidative stress may be allowing the cells to grow faster. But then when you treated with carboplatin, you got an even more significant reduction in the growth of the cells suggesting that TRIP-A1 was protecting the cells from carboplatin induced cell death. And then also you see an increase in Caspace-3 in this window of time at the time that they were harvested. So this supports the idea that TRIP-A1 and vivo could also be protective, not only in terms of tumor growth but also in the context of carboplatin. And I just show you this, it wasn't a very meaningful experiment. He also took the PDX model that had high levels of TRIP-A1. It turns out that they're very sensitive to carboplatin. So this is carboplatin alone. And there was a reduction. It was statistically significant because he used a lot of mice, he used 10 different mice for each of the arms of this experiment. One thing that, so, you know, we really can't draw too many conclusions from this. Oh, in this case, he used the inhibitor of, he wanted to do the experiment with an inhibitor of TRIP-A1. So he used the TRIP-A1 inhibitor. It has a half-life of only two hours. So he had to dose several times a day and it was a really unpleasant experiment to run. So it's too bad that it turns out this tumor is so sensitive. One thing he did notice is that in the combo treatment, there was a lot more, even though the tumor size was similar, there was a lot more stroma and the stroma comes in when you get loss of cells. So suggested that there might be a more significant effect than seen here, but we need to do, first of all, we need much better inhibitors. So we're hoping that drug companies are gonna come up with better ones. And then also, we need to use a model that's less sensitive to carboplatin in order to be able to address this better. Okay, all right, so then how does TRIP-A1 work? How is it allowing survival of cells even in the context of high reactive oxygen? So, Nobu has done, literally, I think thousands of assays for this and I'm gonna spare you most of those. Just gonna show you a few representative slides, but basically we collaborated with Gordon Mills who has this beautiful RPPA platform that allows you to look at about 300 different cellular proteins or phosphoproteins in order to monitor pathway activation. And so Nobu has looked in MCF-10A cells with or without TRIP-A1, with or without H2O2 in 2D culture or with or without suspension culture. And then he took the breast and lung tumor cell lines, incubated them with or without TRIP-A1 hairpin or AP-18 in order to get the full spectrum of pathway activation. And so, again, it's actually getting late. So I'm just going to show you that we do have lots of data and it was very robust, the data, because we saw very similar effects under all those different conditions, either gain of function or loss of function, which implicated multiple signaling pathways downstream that were activated by TRIP-A1 under conditions which it was specifically activated by oxidative stress. And so you can see here, there's activation of both the IRC and the PI3 kinase pathway. When he just here visualized all the pro and anti-apoptotic proteins, you can see that the strongest up-regulation of anti-apoptotic proteins were MCL1. We also sell BCLXL up-regulated as well. And again, you can see it under conditions of H2O2 or detached. And the way he visualized this data is to show the ratio, this is the ratio of the antibody signal in the H2O-treated cells in TRIP-A1 expressors versus not. This isn't the signal with H2O2 alone. It's relative to the MCF-10A cells that aren't expressing TRIP-A1. And then in parallel, he did the knockdown experiments. And again, the data supported the loss of activation of the same pathways that were activated by overexpression of MCF-10A of TRIP-A1. And you can see here that both MCL1 and BCLXL were down under conditions in which TRIP-A1 was knocked down in either the breast or the lung tumor cell lines. And just one experiment that Nobu did was to address the role of MCL1. And you can actually just look here. So in the control cells, if you just use a MCL1 specific inhibitor, does that affect the survival of the cells in the center? And you can see these are in the, this is in the HCC1569 cells that you lose the inner cells under conditions in which you inhibit MCL1. And these are just the TRIP-A1 controls where you also lose it. And this is the quantification. So MCL1 does appear to be a critical mediator of survival of these cells in the center. Okay, so this just kind of summarizes what he found. I didn't go over all this data. But what he sees is an increase in GTP loading of RAS, which is consistent with seeing both the ERC and the PI3 kinase, an mTOR pathway activated. And I'm just showing, this is like, this is just to impress you. He did lots of assays. Not only did he do this in culture, but recently in order to address the reviewers question whether this happens in vivo, he did it on all the tumor cells. So I have an equal number of panels from all the tumor cells showing this pathway. Okay, so how does activation of TRIP or calcium activate the RAS pathway? And through a lot of experiments with knockdown inhibitors, et cetera, which I have a whole another set of slides that I'm not gonna show you. It turns out that camodulin is essential as well as PIC2. And it turns out that PIC2 in the firm domain, which says this is the closest relative to FAC, the focal adhesion kinase, it has a firm domain that binds to camodulin. And the FAC, its closest relative does not. So PIC2 is activated by calcium camodulin. And then this leads to activation of these pathways in a very significant way, leading to MCL1 activation. So the premise is that the reason why these cells can survive in the insult of ROS is because of strong anti-apoptotic pathways that are activated by calcium. And just kind of as an aside, and I didn't have, I knew I wouldn't have time to go into this, that I think in general, the cancer field of cancer ignores calcium to a great extent, not everyone. There are papers on it, but there's reason to believe that because of data like this, that influx of calcium can have a really strong anti-apoptotic and likely contributes to the survival of cancer cells in ways that really haven't been explored to the extent that they should be. Okay, so basically, just to summarize, there are multiple, as I mentioned, multiple pathways that lead to the induction of ROS. And so what we know from previous work is that most of the antioxidant programs function by neutralizing ROS. And we just are distinguishing the TRIP-A1 protection from ROS because in this case, we're not neutralizing ROS, but it's allowing the cells to tolerate ROS. So as I mentioned before, what we found recently, and again, I couldn't have time to add it, is that TRIP-A1 is activated by NRF2, so that would indicate NRF2 is both neutralizing ROS as well as cleaning up by actually providing an additional mechanism of allowing the cells to tolerate any ROS that's not neutralized. Okay, so just like, what are the therapeutic implications of this? So these data suggest that counteracting antioxidants can enhance therapies associated with ROS. So many different therapies lead to an increase in level of ROS. And so resistance to these mechanisms have been shown to and probably more likely than we realize are associated with upregulation of antioxidants. So could targeting antioxidants increase the sensitivity to these standard of care reagents? So how would you do that? And so we would suggest that potentially one mechanism would be to inhibit TRIP-A1, and we're hoping that these inhibitors of TRIP-A1 have better pharmacokinetic properties so that they could be more effective in treatment. We're talking to radiation oncologists because we think radiation might be a really good approach to start with because you wouldn't have the combined effects of TRIP-A1 inhibition plus chemotherapy, but potentially sensitize the cells without significant systemic effects of chemotherapy. So we think that would be an interesting approach to look at. Clearly lots of people now are looking for ways of suppressing the antioxidant consequences of NRF2, both in tumors that have mutations in the NRF2 axis as well as other tumors that upregulate NRF2 as a result of other like RAS activation. And then there's some really interesting data from Stewart-Triber's lab in a group at McMann's group at UCSF, suggesting that the antioxidant GPX4, which specifically neutralizes lipid proxidase, may be a critical enzyme to maintain the viability of mesenchymal persister cells that persist after treatment with a whole variety of different targeted and chemotherapies. And then a lot of people are looking at blocking glutathione production by inhibiting the critical, great limiting step in glutathione production. And then lastly, I'm sorry for being over, just important consideration in all this is that while antioxidants may promote cancer, they also are important in preventing aging and other associated diseases. So obviously one has to find, as we have to do with all, cancer therapies find the right balance. And we would propose that it's really critical to understand the mechanisms involved in antioxidant promotion of cancer and inhibition of aging phenotypes so that we could find more specific mechanisms that would potentially not, in which we wouldn't have the secondary consequences of inhibition of antioxidants with aging and other associated diseases. We think for a one might be one such mechanism. And then I just like again to acknowledge Nobu who was just, he's the real hero of this. He works seven days a week, 16 hours a day. It doesn't get to see that little guy I think very often. And several people in our lab actually contributed to these studies and we have important collaborations with all these people. I think I mentioned a lot of them, most especially Gordon Mills because of RPPA was so critical in identifying the pathway that was responsible for this and acknowledged funding from several different organizations. Thank you very much. Well thank you Joan for that really terrific talk and we got an extra bonus of a few extra minutes too which is always great, you know. Thanks for making me feel like that. Well, I'm guilty of that myself sometimes as some of the audience knows. So in any case, just as a token of our appreciation of your giving this fantastic talk, we have this plaque to bestow upon you. This falls within all government requirements in terms of the value. It's from a sentimental point of view extremely valuable but it falls within all guidelines and monitoring. I promise not to eat it. So I know you can't give me food. So anyway. Thank you very much. Yes, thank you very much. All right, and so I think we have a little bit of time for some questions. Do we have any questions from the group? Yeah, yeah. Alejandro. The TRP-8-1 channel is open and excess calcium ions enter the cell. Is there a secondary effect on calcium movement across the endoplasmic reticulum memory? You know, we can't see that with fear too so and we haven't explored it. So I can't answer that question specifically. I know that if you give really high concentrations of AITC that it will go into the mitochondria and you'll get death. So, you know, typically it's self-limited. You get these oscillations but if you use too high a concentration well but I can't, and we haven't followed the ER yet. Are there any other calcium channels that my work in conjunctions with the TRP-8-1? So if you, you know, some of those first slides there are other trip channels that are activated or that are overexpressed. We looked at TRP-M and we don't see any activation under the conditions that we've looked at but I, you know, there's so many different stimulants of those different receptors and there's a couple of papers in the literature suggesting that there can be survival benefit from others but mechanisms haven't been worked out yet. So, and then there's, you know, TRP, I mean not trip channels but muscarinic receptors are overexpressed in lung cancer and stimulated by acetylcholine and acetylcholine is produced in the tumors and there's evidence that vagal nerve stimulation can be protective in the gut tumors. So I think there's a lot more out there that needs to be learned. Yeah, it's really interesting to talk to you on. So it looks like TRP-8-1 has a sort of outsized function in 3D cultures and that's probably why it might have been missed by a lot of the regular screens in 2Ds. So in this context, do you see a synergistic effect of TRP-8-1 inhibitor with arc inhibitor, mac inhibitor, for example, in the KRAS mutant lung cancer or with PI-3 kinase inhibitor in PIX-CSU-A or P-10 mutant tumor cells? So we haven't looked at that. Although I think since we get total clearance when we knock down TRP-8-1 or inhibit it, I think that we would see that but we would accelerate the clearance, I think. We probably should do that. I think that we just have to do it temporarily because we do end up with hollow spheres. But I think, I bet we would because that would be eliminating one of the pathways that's most protective. We also haven't looked at which pathway controls MCL-1. In the literature, it looks like PI-3 kinase controls MCL-1 so that would be predicted. Other questions? So I have a question which may be totally off the wall but I'll ask it anyway. And that is, have you looked at all in terms of gas-dermin mediated cell death in these systems? And the reason I raise that question is that there's at least some evidence that cisplatinum may act to kill cells by activating, I think it's gas-dermin E rather than gas-dermin D, which can be activated by the inflammasome, which can be activated by reactive oxygen species. So I'm just, you know, trying to put these under my... The whole loop. Yeah, trying to put these things together. I mean, we don't have to implicate anything else because those cysteines themselves are reactive but that's not to say that there couldn't be something else that would contribute as well. Well, I'm thinking of the gas-dermin as being the executioner of the cell. You know, so that you'd have... Oh, when we knocked down tripe-1. Yeah, yeah, yeah. Yeah, well, actually, I never thought about it. We could look at that. Yeah, I mean, it's an interesting... Feng Chao has been working on the gas-dermins and, you know, the role of gas-dermins in cell death from chemotherapy. And, you know, it's just, there's some things of what you talked about that, you know, sort of fit into that. Yeah, I'm sure we should look at that. Yeah, that's interesting. Yeah. Oh. Okay, other questions? All right, if not, thank you so, so much. Thank you for coming.