The Naked Truth about TGF-α

we’ll get afternoon I’d like to welcome everybody to the next installment of the Vanderbilt discovery lecture series my name is Rick peek I’m the division of Gastroenterology chairman and it’s my distinct honor and pleasure to introduce our speaker today dr. Bob coffee Bob is professor of medicine in cell and developmental biology who’s also an Ingram professor of cancer research and I found out this week that Bob’s mother and sister are here so spent a very long time coming up with a glowing and all-encompassing introduction and Bob called me yesterday and he said he had a lot of slides and if I did that he’d have to resort to east cuts tactics that he learned during his training and he was going to break my knees so I’ve been given two sentences that I can say about Bob so Bob did his internal medicine training at Emory he then did dual fellowships at Mayo and Georgetown and oncology and gastroenterology and he joined the faculty here at Vanderbilt in 1986 as assistant professor secondly Bob has been an outstanding role model for physician scientist ever since he’s gotten here and many of the lessons that I’ve learned as I’ve moved forward have come from Bob and his successes and his research really is focused on EGF receptor signaling TGF alpha signaling and how they influence both disease and normal biology this lecture today and over the next 50 minutes I’d like to tell you about my 20 year journey in in research and so my nephew Hayley Proctor is graduating from the underground undergraduate campus tomorrow and as a result my sister and Proctor here today so I wanted to describe this journey in a way they can understand and also help my mother discover what it is I’ve been doing in the lab rather than how I seen patients so as part of my GI fellowship at the Mayo Clinic in the mid-1980s I was expected to do a year of research and so with absolutely no lab experience I was very fortunate that hal moses accepted me into his lab and much to my surprise i became totally enthralled by the research enterprise the process of posing questions performing experiments formulating testable hypotheses based on the results and then performing more refined experiments so I found this iterative process of discovery addicting and have been spent over 80% of my time over the last 20 years doing lab based research and the research has had a translational thrust based on my clinical training and how Moses discovery lecture two weeks ago he provided a historical perspective on transforming growth factor alpha and beta initially thought to represent one activity isolated from the condition medium of virally transformed cells and able to transform normal fibroblasts it was really realized at that time in the early mid-1980s by house and Mike’s porns lab that there were actually two activities TGF alpha and tgf-beta representing two distinct gene products bound to two distinct receptor systems moreover when the two proteins were added individually to epithelial cells TGF alpha stimulated growth whereas tgf-beta was a growth potent growth inhibitor so that’s where things really stood when I entered house lab being interested in colon cancer I decided to characterize TGF alpha and tgf-beta production and human colorectal cancer cell lines as control I obtained RNA from normal human keratinocytes to look at gene expression by northern blood since it was thought at that time TGF alpha and tgf-beta were only made by cancer cells it seemed reasonable to think that these normal epithelial cells would be a good negative control amazingly I found that TGF alpha was made by these normal epithelial cells and when TGF alpha protein was added to these cells it induced increased expression of TGF alpha mRNA so this was an example of a feed-forward process of auto induction about that time house lab found that tgf-beta was also produced by normal cells this then led us to formulate a model in which TGF alpha was produced by these cells and acted in an autocrine matter to stimulate growth TGF alpha was also produced by these cells but in an inactive state so upon its activation tgf-beta inhibitory effects would override the stimulatory effects of TGF

alpha we predicted that a feature of the malignant process might be a loss of the growth inhibitory effects of tgf-beta signaling and accentuated TGF alpha growth stimulatory signaling and time has borne out this prediction to be true so at that point how decided to take the job as chairman of cell biology at Vanderbilt and he asked me to join him there were good opportunities for my wife Murray Griffin and myself so we decided to accept the position at Vanderbilt so when we left Rochester in January 1986 it was minus 40 and when we arrived in Nashville it was 40 degrees so with this eighty degree temperature swing Murray thought that we had followed Moses to the promised land so here is Stan Cohen receiving the Nobel Prize in 1986 for his discovery of EGF and the EGF receptor when I first visited Vanderbilt I met with Stan and asked him whether a person like myself trained in clinical medicine could contribute to basic science he cupped his hands over his eyes and said yes if two things happened if you pay careful attention to your data and you’re lucky so I think that was good advice and I’ve been very fortunate to be able to work at Vanderbilt and it’s largely collegial environment and to be associated with so many talented and dedicated individuals in my lab and interact with so many wonderful colleagues at Vanderbilt so much of my research has revolved around studying roles for TGF alpha and normal physiology and its possible role when overexpressed and contributing to cancer the first thing I learned to appreciate was that life was more complicated than I thought so there are actually seven mammalian ligands that bind to the EGF receptor EGF TGF alpha and fo regulan Epogen beta cell EO and heparin binding EGF light growth factor and epi regulan so upon binding of a growth factor to the receptor the receptor can homodimer eyes or it can partner with three other EGF receptor like molecules that can increase the diversity of signaling of signals that are being transmitted after growth factor binding now all seven of these ligands are produced as transmembrane proteins so they’re inserted into the plasma membrane whereupon they’re cleaved by cell surface pro users to release the soluble ligand that then can bind to the EGF receptor and I’ll be telling you more about TGF alpha and aunt 4e and ampere regulan as we go along now we found sizable amounts of T in the stomach and the colon and so studying roles for TGF alpha in the stomach led us to implicate it’s overproduction in the pathogenesis of Menezes disease so now what in the world is Menezes disease so it’s a rare acquired overgrowth state of the stomach these patients present with a fascinating constellation of signs and symptoms they have severe oftentimes refractory nausea and vomiting abdominal pain they produce little asset because parietal cells to sell in a stomach that makes acid is strikingly reduced in this condition if not absence and and they selectively leave protein across their gastric lining and so the loss of the protein from the vascular space reduces the oncotic pressure and results in severe peripheral edema oftentimes even an Asarco now in the adult it’s a progressive disorder there’s been no effective medical therapy for this condition and patients oftentimes come to total or subtotal gastrectomy because of refractory symptoms or concern about malignant degeneration and the etiology is felt to be uncertain so here is a picture of a specimen a patient with Menezes disease and the stomach is normally four to five times thicker than normal and Menezes himself described these as cerebral convolutions which i think you can appreciate and and then this is showing that disorganized gastric mucosa

there’s cyst formation it bears little resemblance to what we would consider normal body and funda histology which I’ve depicted here so normally you see this very ordered arrangement where there are surface mucous cells that extend very briefly or into a short into the stomach and then the glandular portion of the fundus and body of the stomach which is made up of predominantly parietal cells and chief cells and then this is a 3d reconstruction of the normal stomach showing these surface mucus cells and these pitted out pouches which are the parietal cell the acid producing cell in the stomach now these patients they have severe increased mucus production thick folds you can see a thick mucous bridge at the time of upper endoscopy and Phillip Manette Ria’s first described this entity in 1888 in the journal Archives gives physiology and at that point the editors of the journal were brown support and and Charcot and so those of you familiar with Browns cord syndrome and church arco Marie tooth disease will realize that eponyms were really much in fashion at that time so these were really the good old days when if you described a disease or defying two syndrome you just named it after yourself so here’s the original drawing and this this volume actually exists in the Vanderbilt Medical Library of Philip Manette rias showing the gastric histology associated with Manette Ray’s disease and he did a wonderful job of showing the marked surface Foley / hyperplasia of the surface mucus cells they have extended deep into the gland and there’s a marked reduction in the number of parietal cells and chief cells and two of the four cases that he described and that year had gastric cancer so the antral involvement and the link with gastric cancer are features of the disease that continue to this day so this just gives you a another representation of the stomach so in this disorder it normally involves the body and the fundus of the stomach the proximal portion of the stomach and spares the antrum so what’s the link between TGF alpha and Menezes disease so having found TGF alpha in normal epithelial cells we saw that there was quite a bit of it in the stomach and then when we began to look at where it was localized by immunohistochemical staining for TGF alpha we could see that we saw some staining at the surface at the surface very surface of these cervical cells and then weren’t prominent staining in the parietal cells and I need to tell you at this point that what we’re calling a normal stomach is really the stomach of either a young Dan Beecham or myself so back in those days we would take turns being endoscope and harvesting tissue that would then be considered normal I suspect that today this would be considered a HIPAA violation so then we conducted about 10 years of research understanding roles for TGF alpha in the stomach and showed that it could stimulate epithelial growth gastric epithelial growth it could inhibit acid it increased mucin and then in separate studies that we have done in collaboration with Brigitte Hogan we had shown that sustained overproduction of T V F alpha in the mouse mammary gland resulted in breast cancer so based on those pieces of evidence and knowing these features of metree age disease we speculated that perhaps overproduction of TGF alpha might contribute to the pathogenesis of Menezes disease so we then looked at 17 patients and look for TGF alpha amino staining in the stomach and we could see that there was a marked increase in TGF alpha immuno reactivity in these markedly expanded surface mucous cells these fo v ol or hyperplasia and we then turned to mice

that overproduced TGF alpha in the stomach and saw that they had all of the histological and biochemical features of Menezes disease so I only have time to show you one feature of that but we can see here so these are non transgenic littermates the metallicy named TGF alpha mice this is a zinc inducible promoter so after a month we put the mice for one link one week on 50 millimolar a zinc sulfate and then examine the stomach’s and and both mice and we can see even after this short period of time that there’s fovea lor hyperplasia we’re using here now an antibody that’s selective for the parietal cells and we can see that there’s a significant drop-off in the number of parietal cells and there was also a significant reduction in basal and histamine stimulated acid production in these mice now we and and these studies were done in collaboration with Jim golden ring we then carefully studied these mice over time with cereal pulses of bromodeoxyuridine and looked at various markers of proliferation and apoptosis various lineage markers and we were able to determine that the poloidal cells in chief cells that were markedly decreased were not decreased because they were being destroyed it appears that they weren’t being made and so we put forth a model that in menezes disease there is a selective expansion of the there’s an expansion of the progenitor zones and a selective increase in the surface mucous cell population at the expense of parietal cells and chief cells so we hypothesized that this was due to increased EGF receptor signaling and we had also found in several the patients that we had looked at and in the mice that there was a normal serum gasolin which I submit to you is abnormal so gastric acidity is a potent stimulus full gas from production so if you’re not making acid which these mice and these patients aren’t you would expect the gastrin to be very high but in point of fact in these situations it’s normal so our model was increased EGFR inappropriately normal gastrin and so this really set the stage for us to be able to obtain compassionate use approval from the FDA to treat a patient with Menezes disease with a monoclonal antibody that blocks TGF alpha from binding to the receptor so this patient had been referred to us from UT Southwestern was severe nausea vomiting and edema from Manetti EA’s disease he had failed all medical therapy and was unable to undergo surgical remove removal of his stomach so we then turned to suck some a vol so-called C 2 2 5 or Erbitux monoclonal antibody that blocks the ecto domain of the EGF receptor such that the ligand cannot bind now some of you will be familiar with this drug because this is the drug that got Martha Stewart in so much trouble trouble for insider trading so Martha ended up in jail for for the insider training and and this is a reference to sell not the reference to sells that we normally think about but nevertheless we we utilize this drug on a compassionate use basis and so the the drug was administered in a loading dose of 400 milligrams per meter squared intravenously and then we gave him three subsequent weekly doses and then we objectively analyzed the results I should say that within several hours he was feeling much much better but we were obviously concerned that this could just be placebo control but when we then looked more carefully in the gastric mucosa so because of the thickness of the stomach we could take biopsies before treatment one day after treatment and then one month after treatment and when we did that and then here looked at proliferative indices in his stomach we could see that there was no change in total map kinase but within 24 hours of treatment there was a plummeting of active phosphorylated map kinase and

that correlated very nicely with ki-67 staining this is a marker of cells that are proliferating and we can see that there were 45 positive cells per glandular you know pretreatment but in multiple fields that K Washington counted we had on average of a significant reduction with five positive cells per glandular unit with just within one day of administration and so then when we formally studied him after one month we could see that vomiting had gone down significantly so we had a grateful patient on our hands the serum albumin had gone up and the stool protein loss had sittin gone down significantly and now there were parietal cells so this is a pretreatment biopsy here standing for that selective marker for parietal cells no staining but then after one month we could see the magenta colored staining indicating parietal cells now we normally think that there’s coordinate regulation of both map kinase and a KT so activated phosphorylated map kinase conferring proliferation active phosphorylated a KP contributing to survival so when we activate the receptor we normally think both of these parameters will be upregulated when we block the receptor we think that they’ll be coordinate ly down regulated but when we looked in this patient one day we found an increase in total and phospho a keiki and then when we looked at one month we could see that the phospho a KT was Co localizing with these parietal cell specific markers so it appears that perhaps we’re seeing the signals emanating from two different cell types and it’s possible that this survival signal is turned on to ensure that the now emergent parietal cells survive this also gave us an opportunity to test our hypothesis that there’s increased EGF receptor signaling in the setting of an inappropriately normal serum Gaffin so this patient had a pH of 7 in his stomach normal people would have a guess of pH of 2 or 3 that should be a profound stimulus to increase your serum gastrin but in point of fact with that gastric pH his serum gastrin was a t less than a hundred being normal after one day it’s well over 400 and at 1 month or sustained up regulation of the gastrin to 591 so it’s known that gastrin is Kovach factor for parietal cells and for chief cells so at least based on the careful study of this patient we could determine that our model it was still intact that in fact it may be that in Manette reaiized disease there’s this expansion of the surface mucous cell compartment at the expense of parietal cells in chief cells and that when we blocked the EGF receptor and see a increase in serum gas gastrin we can restore a more normal lineage allocation in the stomach so this successful treatment then led us to develop an fda-approved clinical trial we’ve treated five additional patients successfully through this trial and two additional patients are too soon to enter the trial so the trial that was conducted was really spearheaded by a very talented MD PhD student in the lab Steve settle Steve’s now a radiation in radiation oncology training at MD Anderson Kevin Finnegan another GI fellow has picked up on this project and he’ll be presenting those results at an oral presentation at the aga later this month hailey Fisk the new GI fellow is is now very involved in continuing this trial and we’ve had ongoing and very important collaborations with Chris Lindh and Kay Washington so this is just a profile of the patients that we’ve treated so here are the six patients that we’ve treated all of them have had dramatic clinical and biochemical improvement and patients have been coming from from all over the place so this particular patient RI a 79 year old woman a mother of 18 came from Lebanon and this isn’t Lebanon Tennessee this is the country Lebanon and she came shortly before hostilities broke out in that country interestingly four of the patients that we see

have had ulcerative colitis and to my knowledge that link between all sort of colitis and Menezes disease has never been made before this patient was interesting he was referred from Seattle and from the interval of that time that he was diagnosed to coming to Vanderbilt his symptoms improved and he had spontaneous resolution of Minette rage disease and we think that this is the first adult case of CMV induced Menezes disease so in the interest of time I’m only going to give you two examples of the kind of responses that we’re getting treating these patients now so here’s a lady that came to us from UCLA and at UCLA she had been on periodic albumin infusions and PPN to keep her albumin at a reasonable level they had bolus tour just before she came to Vanderbilt but then once we started c2 to five we could stop the albumin infusions discontinue the TPN and now with increasing lengthening intervals of treatment we’ve been able to treat her now well over two years and been able to maintain her serum albumin at a reasonable level we’ve gotten some insights into the protein loss so here we’re adding a radio-opaque dye ruthenium red and we can see that pretreatment in a ex-vivo harvested piece of stomach that this dye is able to work its way down along the para cellular route and that once we in introduced treatment within 24 hours to die now is retained at the cell surface so these junctions have have tightened very dramatically and very promptly and this persists at one month here’s another patient that we recently treated so this is a gentleman who came from the University of Pennsylvania pretreatment and you can see by CT scan these thick his gastric mucosa and then after four weekly injections you can see there’s just a thin rim of normal gastric mucosa here of the gastric lining we can see histologically that here’s the marked foveal or hyperplasia with a significant reduction in parietal and chief cells and after one month you can see that we’ve restored the stomach to a much more normal appearance the foveal or hyperplasia being retained to the at the surface and now a marked increase in the number of parietal cells and these parietal cells are working because his pH before treatment was 7 and after a month it’s 2 so by my calculations that’s about a hundred thousand fold change in gastric acidity over that period of time and here is evidence of no parietal cells and then a marked increase as shown by this selective parietal cell marker or hydramat aciem 8 EPA’s so I want to make the point that there is merit in studying a rare disease so it was stated most eloquently by William Harvey in a letter to a dutch physician two weeks before he died and he said at that point nature is nowhere nowhere more accustomed to openly display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of nature by careful investigation of rarer forms of disease so I’d like to make three points I think we’ve gained new insights into the pathogenesis of this disorder this work provides an example of how when we begin to understand the pathogenesis of a disease we can fashion effective therapies and the work has also helped us to better under to better monitor colorectal cancer patients who receive so Topsham app since this agent has been approved by the FDA for the treatment of advanced colorectal cancer a final point is that this work could not have been accomplished without the support of the co of the clinical research center and Dave Robertson Linda Lane and their superb staff and here are two individuals from the clinical research standard Krista heads from Atlanta

Howard who were instrumental in the successful conduct of these studies so we’re planning to treat four additional patients to complete the trial obtain FDA approval for Erbitux as the treatment of choice for Menezes disease and we have much more work to more fully understand a number of the features of this disease that I think were uniquely positioned to be able to accomplish so now I’d like to shift gears and frame the remainder of the talk from the perspective of a three prong paradigm we’ve established to study colorectal cancer so we try to move seamlessly from in vitro systems to in vivo models to propel forward innovative diagnostic and therapeutic approaches to colorectal cancer so we study polarizing epithelial cells in culture to simulate the way our epithelial cells are ordered and function in our body so epithelial cells are at the interface between the external environment and our internal milieu they grow in a polarized manner with the apical surface facing the external environment and the basal lateral surface facing the interior type junctions form a and witness it’s important to recognize that the EGF receptor is confined to the basal lateral surface of polarized epithelial cells so the very simple question that Peter dent in a lab an independent investigator at the University of Michigan the question that Peter asked was to which surface do EGF receptor ligands traffic when they are produced by polarizing epithelial cells so the major premise of this work is that the spatial and temporal organization of proteins in the context of polarized epithelial cells will provide fundamental insights into epithelial cell biology so for these studies we’ve used the prototypical polarizing epithelial cell M D CK to derived from the dog kidney but we now also study a battery of human colorectal cancer cell lines that we’ve identified that retain the property to form a uniform polarizing trans well while when grown on trans well filters one is then in a position to either be able to add to or sample selectively selectively from the apical or the basal lateral surface we can begin to ask questions where are proteins where lipids directed in the context of a polarized epithelial cell and so this slide briefly summarizes ten years of work that describes four of the seven ligands and the trafficking patterns that four of the different ligands take when they’re produced by a polarizing epithelial cell in the interest of time I’m only going to tell you about two so we know that both TGF alpha and alpha regulan are delivered preferentially to the basal lateral surface they’re both cleaved by the same enzyme called taste also called atom 17 and then soluble ligand is released in the case of TGF alpha it’s avidly taken up by the EGF receptor in the case of ample regulan which has a heparin binding domain in its mature portion that helps distinguish it from PDF alpha it actually binds better to heparin sulfate proteoglycans than it does to the receptor even though it still binds the receptor and it’s thought that all of its biological actions are mediated through binding to the EGF receptor so this is the data showing that TGF alpha is a locally captured growth factor so we could show in these MDC K cells that we had engineered to produce large amounts of TGF alpha and had developed a very sensitive and specific radio immunoassay for TGF alpha that we could not detect TGF alpha in the basal media of these cells that had approximately 40,000 basal lateral EGF receptors until we added the monoclonal antibody to the basal but not apical surface we could see that there is now a marked up regulation of TGF alpha this was we think an important biological observation and we can now thinking if our polarizing cells may have have relevance we can now look in patients that are treated with the monoclonal antibody and here’s one of our Minetti

AIDS patients in which there’s a nearly three-fold and a fully three-fold increase in TGF alpha following 24 hours after the first dose of the monoclonal antibody so because we now know that as soon as TGF alpha gets to the basal lateral surface its cleaved and as soon as it’s plead its gobbled up by the receptor we reason that an important step in understanding how endogenous TGF alpha is regulated was to understand how it gets delivered to the cell surface and we know looking across species the most conserved portion of TGF alpha is this 39 amino acid cytoplasmic tail the last three residues are a PD Z recognition motif we’ve identified a number of pdz containing proteins that bind there but that interaction influences the efficiency of trafficking but does not impact on the fidelity of trafficking so we can remove those four residues TGF alpha still just goes to the basal lateral surface it just takes longer for it to get there so Quincy Lee in the lab then made a yeast two-hybrid library from one of our polarizing colorectal cancer cell lines he had identified that basal lateral sorting motifs resided in these two domains in the tail and using that yeast two-hybrid library and using the tail as a bait he identified that naked – but not naked one was able to interact with the basal lateral sorting motifs in the cytoplasmic tail of TGF alpha now this assumed great interest to us because we knew that Matt Scott at Stanford had cloned naked cuticle a year or two later naked 1 and naked 2 were identified and Matt in the fly Drosophila had been able to show genetically that naked cuticle was an inducible negative regulator of went signaling so upon activation of the canonical went pathway not only were jeans like Mick and maitre licen upregulated but naked cuticle was up regulated which in turn binds and in activates dishevelled a positive regulator of canonical in signaling so here we had a possible convergence between EGF receptor related events and when signaling which we know is critically important in the pathogenesis of colorectal cancer so Quincy over the last several years has identified a number of motifs in this 451 amino acid protein previous to this work there had been no biochemistry performed on this family of proteins and what Quincy has been able to show is that naked 2 is required for the efficient delivery of you have alpha to the basal lateral surface so here depicted is the TGF alpha tail binding domain of naked 2 which interacts with the cytoplasmic tail determinants another very important observation that Kunze made was that the second residue of glycine undergoes a post translational modification it undergoes Marrissa latian and proteins that are marissa lated are able to bind to membranes and so what kanji was able to do was make a single base pair of change and convert the glycine to an alanine now naked 2 is no longer Marisa lated and now let’s look at what happens to the localization of naked 2 gfp we can see in these polarized MDC k cells wild-type naked tubes along the lateral membrane and in the g2 a there is a asymmetric clump of gfp and we can show that in the g2 a naked 2 expressing cells we see an accumulation of vesicles at one corner of a polarized epithelial cell so the model that we’ve been able to test and I think verify is that make it to interacts with a golgi processed form of TGF alpha so TGF alpha containing vesicles when they bud from the trans-golgi at that point and not before make it to recognize as the cytoplasmic tail of TGF alpha it coats those vesicles it has all of the information to be able to deliver those vesicles to a corner so these vesicles go directly to this corner of a

polarized epithelial cell where the vesicles now dock and fuse in a naked – Marissa Latian dependent manner so these vesicles are very dynamic in the wild-type state there they kiss and run they touch and fuse and then they fuse at the membrane g2 a marissa Latian deficient make it two expressing cells those vesicles exhibit no social behavior there there they don’t talk to one another and they don’t fuse at the membrane so we presumed that the worst elation deficient naked to expressing cells that the GTA was acting in a dominant negative fashion and here clumsy is verifying that by knocking down with shRNA naked too and can show that there’s now an accumulation of cytosol ik TGF alpha and we see now no cell surface TGF alpha when we knocked down endogenous naked 2 in these cells now these effects are very specific for TGF alpha so there isn’t a global effect on proteins that are trafficked to the base of lateral surface and Amarista Latian deficient naked t-cell so here is a very nice control i think so here’s an for regulan another EGF receptor ligand delivered to the same compartment and it gets to the basal lateral surface just fine in wild-type or these marissa Latian deficient make it 2 expressing cells as does sodium potassium ATPase and eek adherent so i want to briefly tell you about work of waiting a very talented graduate students away has done a back recovering back recombinator conditional knockout of naked to those mice are just coming online in the meantime in his spare time he’s been studying the regulation of naked 2 and what he’s been able to show is that naked wang is a target of when signaling and up regulated in most colon cancers whereas naked 2 is down regulated in 60 to 70 percent of colorectal cancers and its losses of late event it doesn’t occur at the adenoma stage it occurs at the cancer stage and likewise he’s been able to show that there is an EGF receptor independent up regulation of naked 2 by t VF alpha so TGF alpha endogenously produced TGF alpha protects naked 2 from ubiquitin mediated proteasome ‘old aggradation to our knowledge is the fur example of a cargo regulating its code so I now want to just touch on some future directions that were undertaking both as relates to the epithelial biology program into our GI Spore so from the epithelial biology standpoint no one has been able to purify basa laterally targeted X acidic vesicles because their transient there are ephemeral their low abundance and so we’ve been able to Jenny Cal I should say in the lab has been able to exploit the fact that the Marissa Latian deficient g2a naked2 expressing cells have vesicles trapped at the corner of the cell these vesicles can’t fuse as depicted here and so what she’s been able to do is be able to do a biochemical enrichment of fractions over anaya docks and all gradient of cells that are either expressing wild-type naked2 egfp or g2a native to egfp and you can see that the position of naked 2 shifts in the gradient whether the cells are producing wild-type or these more wrist elation deficient make it to there is ad enrichment for the g2 a pool to four other membrane other cellular organelle markers so based on that Jenny and working with Jim Higginbotham was able to flow sort these vesicles using GFP and a nonspecific lipid I and now have 99% pure basal laterally targeted X acidic vesicles in collaboration with Amy ham and in the proteomics core we could show that first of all that these will in fact were vesicles from these flow sorted material and we’ve been able to identify 222 proteins that make up this subset of basal laterally targeted EXO Civic vesicles so the way we’re headed now is

Jim Higginbotham is designing a custom flow cytometry uniquely equipped to be able to isolate your cellular organelle of choice and that we think now we can use the flow sorting with different probes and then investigator investigators at Vanderbilt will have available to them the ability to identify these subpopulations of vesicles scattered throughout the cell and then carry out proteomic analysis and the prediction is that over the next ten years we will find that a number of disease states are due to altered vesicle trafficking and I think at Vanderbilt will be uniquely positioned to conduct some significant research in this area and then I want to just close with telling you about the work that we’ve been doing within the context of the GI Spore is for as far as optimizing EGF receptor blockade so we found out recently that our G is for was renewed it received the top score in the country Hopkins was second and Harvard was a distant third so we’re very pleased about that and we’ve got a wonderful team of investigators working on various aspects of colorectal cancer one part that mace Rothenberg and a pill merchant and myself and many others are involved in is optimizing treatment of the EGF receptor axis which we define as the proximal steps and activation of the EGF receptor including ligand cleavage ligand uptake and tyrosine kinase activity we conducted a trial with a wrestler that was not successful but we’re now combining tyrosine kinase inhibition and monoclonal antibody treatment and there’s preliminary work from the group in Spain that they are now getting 56% response rates when they’re combining agents that will hit these two steps in the EGF receptor active axis in patients with colorectal cancer so we’re we’re optimistic about this approach now I’ve tried to highlight the number of people that were involved in various aspects of the Menezes disease David Paige was a very important contributor early on in the studies we have a wonderful group of investigators that have looked at this from many different aspects I I don’t have time to acknowledge all of the people that are presently in the lab the remarkable number of collaborators we have for example Kari van ray is a shared postdoc between myself and we lisanna Koch Russell and Canaries looking at naked one make it to regulation and zebrafish so before I an and though I do want to acknowledge for the contributions of four women who have really contributed to my work the first two having continued to provide support and the second to have provided motivation so first and foremost is my wife Marie Griffin and secondly Ramona graves deal who has been a superb lab manager and a friend for the past 20 years now the next two individuals have provided motivation for how I approach my work and since sunday is Mother’s Day I think the choices are are timely and and apt so the first is is Mother Teresa so when I finished my residency in internal medicine I felt so out of touch with what was going on in the world that I took a backpack and went around the world and I justified this by working with a medical group in most places that I visited so in Calcutta I had an opportunity to work with Mother Teresa’s group and I spent one evening alone with mother Teresa for some two hours discussing what it meant to be a physician and what it meant to take care of the sick and suffering and I had a little pocket Bible with me at that time and I asked mother Teresa if you might put something in the Bible and so this is what she said I can’t can’t I’ll read it for you it’s not showing up as well as I would like but she said be the sunshine of God’s love and compassion to the sick and suffering and remember to pray the work do it with

Jesus for Jesus to Jesus God bless you mother Teresa February 18 1980 so that was actually a wonderful experience and one that I remember very well and upon reflection I realized that the only other time that I felt I was in the presence of a truly good woman who was sitting with my mom who can continues to inspire me by her grace and spirit and so I think I jumped that but here is Mother Teresa juxtaposed against my mom who is 91 years old and is fortunately able to be with us today so I thank you for your attention I would be happy to entertain any questions that’s right so that’s a really interesting question and in fact that gentleman was in two New England Journal reports he was in our report a single patient successful treatment of Menezes but he was also in the first report of the successful use of prostacyclin for patients with primary pulmonary hypertension and so he was treated in 1993 with prostacyclin and one of the first individuals to get that and that treatment continued to 1998 when we saw him and he had bonafide menezes disease so we we wondered and speculated it at that time might hair the hitherto unrecognized complication of prostacyclin be this overgrowth state of the stomach and and we’ve talked to the pulmonary group here and we’ve just never really made much headway and making that connection but we thought it would be worthwhile at least to screen those patients with the serum albumin and if it was significantly down we might do a stool alpha-1 antitrypsin and proceed from there but we don’t have any other evidence for that association Eric well TGF alpha is certainly a target for for naked so we actually army right so we’ve have 222 proteins and our vesicles now and we’re starting to systematically go through those but the difficulty there is one might complain or might white might query us that we’ve now identified trapped vesicles that we’ve made these are immersed elation deficient they’re trapped in the cytoplasm maybe there are accumulating proteins non-specifically so we’ve gone back in at least a 5 of the proteins we’ve begun to look at there co-localization and wild-type make it to expressing cells and have found that to be the case so we certainly have a motor that interacts with naked 2 myosin those vesicles so the expectation is that there will be a number of proteins that interact would make it to we we think that naked which doesn’t Erica alpha is likely to have its own it has vesicular standing law likely have its own set of cargo unfortunately so far it turns out am for regulan which had been our hope that perhaps a plight interact would make it one and be the naked to equivalent for a pro regulan hasn’t hasn’t panned out your father Coffee is surgery for the scene those are very kind and generous

comments judge thank you very much well it’s a it’s a great question so we’ve only treated two of the four patients that we’ve encountered with ulcerative colitis have actually been in our trial we’re actually having another patient that will be here in two weeks who also has Manette Rage disease in all sort of colitis but the two that we’ve treated so far appear if anything to have gotten better from the standpoint of they’re all sort of colitis we we now while we’ve been following up and getting sequential gastric biopsies we’ve also gotten sequential colonic biopsies but Kay and I haven’t had a chance to systematically go through and see how the ulcerative colitis appears in the presence of seduction mapping is it a possible therapy its popping out time will tell Dan so Danis a very interesting question which I really didn’t have enough time to get into so he’s asking when you lose naked – which we see in 60 to 70 percent of colon cancer actually we think that what TGF alpha really does normally deliver the base level surface cleave taken up it’s a very duck-like interaction – perceptor and that helps them maintain epithelial homeostasis and that when that gets disrupted and one way I could get disrupted is this loss of naked – and there’s net and increase since I saw the TGF alpha which is frequently seen in the setting of cancer that one of the consequences of that is that there is now a relatively unoccupied EGF receptor so TGF alpha is normally the ligand that’s binding to the receptor in the absence of naked 2 there’s the opportunity for other ligands to engage the receptor and for regulan is sitting we think in a depo form in bound in the extracellular matrix it may be processed by metalloproteases we’ve shown recently in a couple of papers that amp regulan but not TGF alpha will cause an EMT like transition so the model we consider is the fact that TGF alpha doesn’t bind the receptor other ligands have a chance those other ligands may contribute to various aspects of tumor progression and those models are are now being tested in the lab dr. Collins so we don’t know the answer to that question yet we know that it is binding the target because we can see an upregulation of TGF alpha in both the serum and gastric juice within 24 hours so we’re fairly confident that that increase reflects the fact that the antibody has a greater affinity for the EGF receptor than the ligand does so we feel pretty good about that we don’t know about the long-term effects on production of ligands and I’m in full disclosure we still don’t really know whether TGF alpha is the only ligand that’s up regulated in the setting of Manette Rios disease but we’re now at a point where we have the biopsies we have the material in hand where we can start to really examine that question and the patients that we’ve treated you