Pathology Grand Rounds February 23, 2023 - Jason Mills, MD, PhD

February 23, 2023

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Common Features of Metaplasia and Tumorigenesis in the GI Tract – by Jason Mills, MD, PhD

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00:00Thank you everyone for joining
00:03our grand rounds today.
00:05It is my honor and great pleasure
00:09to introduce Doctor Jason Mills.
00:12He is a Herman Brown endowed professor
00:15at Baylor College of Medicine,
00:17Chief of Research in the section
00:20of gastroenterology and Hepatology,
00:22and the Co director of Digestive Disease
00:25Center at the Texas Medical Center.
00:28So he graduated summa *** laude from
00:31Washington University in Saint Louis with
00:33double major in Russian and biology, right?
00:38Then he received the MD and PhD
00:41from University of Pennsylvania and
00:43went back to Washu for his anatomic
00:46pathology residency and postdoctoral.
00:50Fellowship there.
00:51And he mentioned that he had a
00:55traumatizing experience with Doctor
00:57Peter Humphrey as pathologist here at Yale.
01:01Now when they were, you know,
01:03signing out a big stack of the
01:05Hershey Springs case in the middle,
01:08they had a, you know,
01:09frozen section and had to leave.
01:11And then when they came back and they
01:13didn't remember which stack half stack
01:15they have reviewed versus they have not.
01:18So they had to go over again the.
01:20Except that's the entire case again.
01:23So they will have a reunion
01:25in the afternoon today.
01:27So you can have some conversation on that,
01:30right?
01:31And actually Jason was my PhD thesis
01:34mentor at Washu and so I have known
01:37him for 18 years now and all my
01:40interest in the GI research came
01:42from him and and he was a positive
01:46influence for me to pursue my career
01:49in pathology on the first day.
01:51When I joined this laboratory,
01:53we sit down on the double headed
01:56microscope and he went over mouse
01:58and human stomach Histology and
02:01encouraged me to become a pathologist.
02:03And at that time I was very negative
02:07because I never thought about becoming a
02:10pathologist during my entire medical school.
02:12But see now I'm a I became a pathologist.
02:16That's how influence influential
02:18he is and as pathologists.
02:21And some researchers here,
02:23we know well what metaplasia looks like,
02:27but we do not know.
02:28Well, you know, how it happens.
02:30What's the process mechanism on the line?
02:33So Doctor Miller says,
02:35focused on his research in the
02:38cellular and molecular process
02:40changes during the metaplasia.
02:42And published more than,
02:44you know,
02:45hundred papers on the metaplasia and in
02:49addition to the seminal scientific works,
02:52he is very talented as he told
02:54you that he majored in Russian,
02:55he is very fluent in Russian and French
02:59and also he can speak some Chinese
03:02and he knows many words in Korean.
03:04So with his talents in language,
03:09he recently coined terminology collagenosis.
03:14Which describes a universal program
03:17how mature cells reenter and change
03:21their subcellular structure and
03:24re-enter cell cycle and becoming a.
03:28The regenerative cells which
03:31happens during the metaplasia,
03:33so he's on the title of his talk today
03:36is the common features of metaplasia
03:39and tumorigenesis in the GI track which
03:42implies the polygenesis basically.
03:44So please join me in welcoming Dr.
03:47Mills.
03:50That's.
03:53Thanks. Thanks so much for the
03:55invitation and thanks to Juan Jay.
03:57I mean it's it's fantastic growth
03:59obviously to come here and and see
04:01people again and meet and meet
04:03new people and but it it's really
04:05fantastic to see you know somebody
04:07that you saw kind of come immediately
04:09can just over from in fact I picked
04:12you up at the airport I think
04:13when you were interviewing for a
04:15Graduate School at Washington St.
04:18Louis and then to have him come
04:19to my lab and then not even be
04:21interested in pathology and coming
04:23to do a PhD and then to wind up.
04:24A pathologist and then here is an
04:26assistant professor and Andre is
04:28the first out of my group to to
04:29become an assistant professor.
04:31So it's just you know it's fantastic
04:34honor and and fun to see see how
04:36things are things grow and and
04:38it was a fantastic introduction
04:39because you know essentially I just
04:41want to I usually don't do a lot
04:44of introduction for how I organize
04:45the talk but because you know I am
04:47a pathologist but also a cell and
04:49developmental biologist my talk
04:51kind of it will go back and forth.
04:53The first part is all going to be sort
04:54of human. Well, it's your background.
04:56And then the middle part is gonna go
04:58all the way down into ribosomes and,
05:00you know, very cell biological.
05:03But if you're interested in human pathology,
05:05don't give up there because it'll
05:06come back also to human pathology.
05:08So that's on the sort of that organization.
05:10And then just as one day said,
05:12as a resident,
05:14I became fascinated with metaplasia.
05:16And, you know,
05:17how do these cells that are sort of
05:20normal cells show up in the wrong
05:22place and how does that happen
05:23at a cell biological?
05:25Point of view,
05:26so that's my clinical interest.
05:27So we we're always doing sort of
05:29translational work on that side,
05:31but then on the research side,
05:33my cell biologist always side says.
05:38You know what what how does cells do that?
05:40How can cells like make this happen?
05:42So what are the mechanisms?
05:43So that's what the talk is about.
05:44So yes the cell biological
05:47changes are the cell,
05:49biological processes polygenesis and
05:51then you know the the context for the.
05:56Pathology is metaplasia.
05:58So.
06:01One just said why don't you put
06:03prognosis in your title so I
06:04I added it last night so.
06:08OK. So you know as far
06:09as I said it's like the,
06:11the clinical drive for this is
06:12how do these metaplasia happen,
06:14how do precancerous lesions arise
06:16along the GI tract and and how
06:18do they progress to tumors.
06:19So that's kind of what drives and
06:22funds our work and lately you know
06:25it's not just us but with the advent
06:28of of single cell RNA seek in in
06:31multiple organs I think you know
06:33we're beginning to realize that
06:35there's a lot more commonality
06:37in metaplasia across multiple.
06:38The Oregon.
06:39So there might be commonality in in
06:41these precancerous lesions that that
06:43you know I was trained when I was an,
06:46you know an AP resident to think it was.
06:51Sort of process.
06:52Interest on metaplasia in the stomach
06:54and and certainly has nothing to
06:55do with how colon cancer starts
06:57or how pancreatic cancer starts.
06:59On the other hand it's kind of like you
07:01know it's kind of becoming clear that
07:03that there's a lot of similarities.
07:05So let's talk about that.
07:07So you know with with Jim
07:08Golden Ring at Vanderbilt,
07:09we had this review recently
07:11in gastroenterology talking
07:13about some of these concepts.
07:15In this one in particular we
07:17focused on the similarities
07:19between Barrett's metaplasia and
07:21gastric and intestinal metaplasia.
07:23And basically you know,
07:24if you think of Barretts,
07:25the ideology there is obviously
07:27quite different from the way you
07:29get intestinal metaplasia in the
07:31stomach and and that's because,
07:33you know, we know that.
07:35Had a thing of of reflux of acid and
07:38and probably also importantly bile as well.
07:41And then that takes your squamous
07:43epithelium right and turns it into
07:45this what in Barretts is called,
07:47you know,
07:48a columnar mucosa at least at first,
07:51which is basically organized
07:53a pyloric gastric unit is.
07:56So it's essentially just a
07:57pyloric metaplasia or a pseudo
07:58pyloric metaplasia from squamous,
08:00although nobody ever calls it
08:01that in the esophagus,
08:03I want to point out that to researchers.
08:05Jim being the one on golden Ring that
08:08coined this term at Vanderbilt that
08:10that that what that means is that
08:12that the cells at the bottom are stem cells,
08:15spasmolytic polypeptide
08:16expressing metaplasia.
08:17And so that's a term that was coined in,
08:20in the stomach actually originally in
08:23humans because spasmolytic polypeptide
08:25is trefoil factor 2 and that shows
08:27up only in these pyloric sort of
08:29lesions in the body of the stomach.
08:31So a lot of what we'll talk about
08:33involves this transition into this.
08:36Mucus secreting deep, antral,
08:38deep pyloric TF2 positive
08:41muck 6 positive lineage.
08:42OK,
08:43so in the stomach,
08:44when H pylori get tired of being
08:45in the Antrim,
08:46they want to expand their knee and they
08:48want to go into the body of the stomach.
08:49And it turns out that the way they do this,
08:51or we can model this with drugs.
08:54And in fact Juan Jade pioneered this.
08:56And there are hundreds of papers now
08:58in the world using this technique,
08:59which is using high doses of tamoxifen can
09:03completely reprogram the stomach of a mouse.
09:06The same way that H pylori can in
09:08humans and actually H pylori and mouse
09:10over a longer time course and what
09:12happens during that reprogramming is
09:14essentially what we talk about as
09:17pathologist as chronic atrophic gastritis,
09:19but is actually also a metaplasia
09:21because it turns the corpus units away
09:24from being oxyntic units with gas with
09:27the parietal cells and chief cells into
09:29basically a pyloric like structure
09:31with MUC 5 AC positive foveolar cells,
09:34a lower ismal.
09:35Proliferative center and again these
09:38deep antral like cells which are
09:41characterized in the stomach as
09:44spasmolytic polypeptide expressing
09:46metaplasia. So.
09:46You know,
09:47the first step basically of H pylori
09:49is to turn normal oxyntic glands into
09:52these pseudo pyloric metaplasia glands,
09:54which is much more what they're
09:55accustomed to in the antrum,
09:56and that's how they spread from the stomach.
10:00So basically what that means is
10:02that that you know as we kind of
10:04learn more and more about Barretts
10:06and we do the single cell RNA seek
10:07and we do the genome studies and we
10:09try to look at the clonal origin,
10:12origin of the Barrett's lesions.
10:14And you know the best consensus
10:17is that these kind of columnar
10:20lesions that look gastric are the
10:21first ones that appear in Barretts.
10:23But even these can be traced back
10:26to roots in in oxyntic mucosa,
10:28in other words, if you do clonal.
10:31Genomic analysis,
10:32you see that these often can be found in
10:34a patient near where they're you know,
10:37most proximal eccentric glands are.
10:40So the idea then is that bile or
10:42acid can turn these oxyntic glands
10:44into these pyloric glands.
10:46And then pretty clearly what happens
10:48then is they become intestinal used.
10:50And why do I say it's pretty clear?
10:52It's because if you look at all Barrett
10:54specimens, especially if you have,
10:55you know, full thickness,
10:56they almost always have bases that
10:58are muck 6 positive or trefoil.
11:00Factor 2 positive or look just like these,
11:03you know spasmolytic polypeptide
11:04expressing metaplasia cells and it's
11:06only the surface at least until you
11:08get a high grade dysplasia that
11:10has a lot of intestinal lization
11:12and of course then the progression
11:14progression from here is into dysplasia.
11:16So our research really is into you know,
11:20how do you get from here to here,
11:21how do you get from here to here
11:22and how do you get from here to
11:24here from a pathology standpoint,
11:25you know then that gives you cancer.
11:27One thing just as a take home
11:29is that we think.
11:31Critical event in all of these
11:33transitions very early on is 53
11:35mutation and we're going to dig right
11:37into the cell biology as we we you
11:39know why we think that now clinically
11:41and Barretts and molecularly we're
11:43finding that that basically as soon as
11:45you have a loss of heterozygosity for
11:47people 53 and and and patients have
11:50loss of function for PD3 then those
11:53Barretts lesions behave differently.
11:55They're almost always become
11:56dysplastic and the rate of conversion
11:59to neoplasms much higher. So.
12:02What I'm saying is that I think,
12:04you know, basically based on
12:06the the lineage tracing and all
12:07these sort of parallels and the
12:09molecular work that we're doing
12:10that what we think happens is,
12:12you know, violent acid comes in,
12:13Barretts and it.
12:15Takes out the squamous epithelium
12:18and then in the in the that sort
12:22of damaged bedding and in that
12:24reflex setting you get migration
12:26of this kind of gastric epithelium.
12:30And then the gastric epithelium
12:33becomes intestinalis.
12:34And so you can kind of see some of
12:36these examples from and a lot of
12:37the work that I was telling you,
12:38the molecular work showing the origins
12:41of the Barretts lesions in in,
12:43you know,
12:44way back at some point in a patient
12:46in oxyntic mucosa is from Stuart
12:48McDonald and Marnick Sanson and
12:50Nick Wright who've been doing this
12:52for a decade or two in in London.
12:54So you can see like these oxyntic
12:57lesions in in sometimes distal
12:59Barretts and then you can see this
13:01is just from their paper actually
13:02and you see these more pyloric.
13:04Regions where you have the spam
13:06mucous cells at the bottom and then
13:08you see spam mucous cells at the
13:10bottom as the tops become intestinalis
13:12used with goblet cells, so.
13:16So we've been working,
13:17we started working on Barretts five
13:19or six years ago and started seeing
13:21that all come together with our stomach work.
13:24And then you know when you kind of
13:26do this sort of thing then you go
13:27back to the stomach and you think again,
13:29well do we really understand how
13:31the stomach metaplasia happens.
13:33And so we started really kind of
13:35digging into the different types
13:37of stomach metaplasia.
13:39You know that from a research side
13:41and and actually in in Asia it's a
13:43diagnostic thing where you really
13:44make a distinction between.
13:46Incomplete and test on that ablation,
13:47complete and test on metaplasia.
13:49In fact,
13:50you know they're type ones and type
13:52twos based on use and patterns.
13:53But what does all that mean?
13:55Well,
13:56it turns out that really if you
13:57go back in the in the stomach and
13:59especially look at the borders of
14:01patches of intestinal metaplasia,
14:02a lot of the times they're they're
14:04incomplete and they have the same
14:06kind of organizations Barretts with
14:08spasmolytic polypeptide expressing
14:09metaplasia type deep pyloric cells at
14:12the bottom and then internalization
14:14of of goblet cells.
14:16At the top and during COVID when
14:18I had more time to kind of mess
14:20around and and look into history of
14:22stuff and I was trying to go back
14:25and and try to figure out where
14:27it was that everybody in the in
14:30the stomach became obsessed with
14:31intestinal metaplasia.
14:32You know is this something that's
14:34always happened because pretty
14:35clearly the first thing that
14:37happens in atrophy is this
14:38more pyloric metaplasia.
14:39Yet we never signed that out.
14:41We never diagnosed that.
14:42I started going back in history and
14:44and you find that people you know.
14:46Have been talking about pyloric
14:48metaplasia actually since like the
14:511890s and it was only in the sort of
14:531960s or 70s that people became so
14:56interested in intestinal medication.
14:58It was about the time that endoscopic
15:00biopsies came around and and
15:02pathologists got only little snippets.
15:04And you couldn't sort of tell the
15:06orientation to tell whether there was basil,
15:08you know, pyloric glands or not.
15:10But even, you know in the 1890s
15:12they kind of had this concept that
15:14there were these sort of pyloric or.
15:16Or acid or mucin cell like glance
15:18at the bottom that these then might
15:20have might be feeding these kind of
15:22incomplete intestinal metaplasia.
15:24This is from a textbook on gastric
15:27pathology in 1897 just to kind
15:30of show this diagram with sort of
15:32spam metaplasia on the bottom and
15:35then internalization on the top.
15:37And then just you know,
15:38I as I do a lot of sort of translational
15:40work and I have slides about my
15:42desk that I look at all the time
15:44and you know bring people in like
15:46Juan J and and sit and look.
15:47You can actually see this pretty
15:49frequently if you look for it where
15:51you can see these kind of deep
15:53pyloric glands erupting into more
15:55superficial transitioning into this
15:56kind of incomplete metaplasia.
15:59OK, so that's stomach and esophagus.
16:02But it turns out now with single
16:04cell or in a site where you can
16:07take apart each one of these cells
16:09during progression to pan in lesions
16:11again for some reason in you know
16:13pathology we only talk about panning,
16:14but in in the mouse where we
16:16can sort of look at each step,
16:18there's an intermediate step called
16:21acinar ductal metaplasia where
16:23the acinar cells shrink and become
16:26more cuboidal columnar cells and
16:29and proliferative.
16:30In an acute or chronic pancreatitis
16:32setting and when you start to
16:34profile those cells by single cell
16:36RNA seek what's interesting and
16:37this was work done at Vanderbilt.
16:40With a from Kathy Delgiorno's group
16:42and and a number of collaborators
16:45including Ken Lau.
16:46I don't think you were on this paper
16:48though on J while you were there, but.
16:51But what you see in these early pancreatic
16:54lesions is the same sorts of gastric cells.
16:57Now of course,
16:58they're not organized into a gland,
16:59you know, they're all on these asinine.
17:01But by single cell RNA seek,
17:02you see cells that look
17:04like foveolar pit cells.
17:05You see cells that look like
17:08these spasmolytic polypeptide
17:09pyloric metaplasia cells.
17:11So and you see the same kinds
17:13of cytokines that are starting
17:14to emerge as being universal.
17:16So I'm not going to talk about this,
17:18but aisle 13,
17:20aisle 33 shows up as mediating
17:23these metaplasia as in the esophagus
17:26and the stomach and even as we're
17:28going to say now in the intestines.
17:30And so the other thing I think
17:32it's been really kind of exploding
17:34in in from the pathology side.
17:36Is that the right sided,
17:38you know serrated sessile.
17:40You know, polyps,
17:42we used to call them serrated
17:44sessile lesions also had this
17:46same kind of basic format.
17:48So in this case you're taking
17:50things that were 100% intestinal
17:52and then now they're moving towards
17:53the gastric side and they wind up
17:55somewhere in the middle with this kind
17:57of pyloric morphology where again
17:59single cell RNA seek shows that.
18:00But then you know,
18:01as I've been collecting these lesions
18:04and we've been looking at them
18:07morphologically and immunohistochemically,
18:09you again see you know and.
18:11And it's been described before too by
18:13others that there's muck 6 positive,
18:15which is exactly the same expression
18:17pattern as spam cells that
18:18emerge that are gastric,
18:20you know,
18:21that are characteristic deep sort
18:23of acinar lesions within these SSL.
18:25And then there's an ad mix sort
18:27of muck 5 AC full Viola and
18:30goblet cell surface lesions.
18:31So at least on the right sided.
18:35SSL type of lesion there seems to be
18:37the same kind of metaplasia but sort of
18:39coming from intestine back towards gastric.
18:42Now that polyps and tubular adenoma
18:43seem to take a different course that's
18:45kind of more traditionally stem cell
18:47based and doesn't fall within that category.
18:50But still now we got four different organs
18:53all converging towards this sort of,
18:55you know pyloric like which is actually
18:58probably maybe one of the primordial
19:00embryonic states of the stomach and
19:02that's probably why and repair the stomach.
19:05Kind of chooses to go back
19:07to this sort of lesion.
19:08But once you have an established
19:11lesion that's mixed lineage where it's,
19:13you know, making both intestinal
19:15and gastric cells at the same time,
19:16you could see at least you know,
19:18reason why that might be a risk
19:21for progressing to cancer.
19:23And so part of that,
19:24you know,
19:25manifest itself when you do genome
19:27sequencing and you look for mutations.
19:29And that's why this is kind of
19:30some of the clinical data for why
19:32people do 3 mutations so important,
19:34which is that,
19:34you know,
19:34in these Barretts glands as they
19:36start to progress and clones
19:37emerge and they start to get the
19:39ones that are mixed intestinal,
19:40it seems like those are the ones that
19:42are prone to developing P53 mutation.
19:44It's those clones that then very rapidly,
19:47you know, from a heterozygote,
19:48once there's a loss of heterozygosity,
19:50they almost immediately go into dysplasia.
19:53And and neoplasia and then and
19:56metastatic and metastasis.
19:58OK.
19:58So that's the like if my talks at sandwich,
20:01that's this is the path introduction
20:03that we're going to delve into what
20:05we think some of the mechanisms are
20:06for how we get these metaplasia and
20:08then we'll come back out again to
20:09see some of the clinical trial work
20:11that we're doing to try to address it.
20:13So the question is to where are all these?
20:18Lesions coming from, you know,
20:19in these four different organs and
20:21you know the knee jerk response that I
20:24would have given you 15 years ago when
20:26Juan Jason the lab was the stem cell.
20:28Everybody thinks stem cells are
20:29what gives rise to, you know,
20:31lesions and and that are proliferative
20:33and gives rise to cancer.
20:34Well, but it turns out, you know,
20:36the stem cells are kind of tricky and
20:38in the pyloric versus oxyntic mucosa.
20:40So the, the professional stem cells
20:42and the oxyntic costs are way up
20:44here close to the surface and
20:45then when you get this you know,
20:47change into this more pyloric.
20:48They're kind of down here.
20:50So there's a change there
20:52already work towards the base.
20:54But then there's another thing that we,
20:56you know,
20:56have to think about which is
20:58that say in the pancreas there
20:59aren't any stem cells at all.
21:00So where are those proliferative
21:02cells coming from?
21:03And and there's been a long strain,
21:05relatively long for this kind of
21:07cell plasticity field of maybe 10-15
21:09years of good mouse work with human
21:11correlation showing that most of the
21:14reparative metaplastic proliferating
21:15proliferating cells in the pancreas
21:17that come about during pancreatitis.
21:19And pancreatic injuries actually
21:20all come from the acinar cells that
21:22are doing their digestive enzyme
21:24secretion that that reprogram.
21:25Well,
21:25it turns out we have a ton of evidence
21:28now that actually similar things
21:29are happening down at the base.
21:31And the reason probably why you get
21:33this change from an oxyntic mucosa,
21:35this kind of organization with
21:37proliferative cells at the base
21:39is because the,
21:40the fuel for these changes in in
21:42these lesions is actually at the
21:44base and the differentiated cells
21:45just as it happens in the pancreas,
21:48in the acinar cells,
21:49it's in the digestive enzymes.
21:50Recruiting chief cells at the base.
21:52So that brings up this concept
21:55that how do you get from a,
21:58a, a differentiated cell,
22:00massive secretory cell like the
22:02pancreatic acinar solar chief cell
22:04to a much smaller proliferating cell.
22:06And you know that actually,
22:08you know stirred us to begin to
22:10explore the idea of cell plasticity,
22:12which is where this fits.
22:14And you know this,
22:15this concept has exploded in the last
22:18five to 10 years and we had the first,
22:20I think the first ever meeting that I helped.
22:23Organized,
22:23which is a keystone meeting in 2019 on it,
22:27but then there was a follow up
22:28and now there are a number of
22:29meetings that are scheduled.
22:30We had a paper on nomenclature,
22:32but just to kind of put us all in the
22:34same cell and developmental biology
22:36page when we're talking about this lesions,
22:38you know the canonical stem
22:40cell idea of how you get.
22:42Differentiation in a tissue is
22:44that you have these stem cells
22:46that make faith choices, right.
22:48And as they differentiate and
22:49they're basically like marbles
22:51rolling down this Waddington,
22:52this Conrad Waddington was
22:54the person who came up with
22:56this concept of a landscape of sort
22:59of differentiation choices and then
23:01the ball sort of slowly roll down
23:03and then you get your chief cells and
23:04parietal cells and acinar cells at the
23:06base and then they just sit there.
23:07You know, the idea inherent to this
23:09concept is that it's a unidirectional
23:10flow of the balls roll down the hill.
23:12And so then if you need to get repair,
23:14any kind of repair done,
23:15then you need to take one of
23:16these progenitors to repair.
23:17But it's pretty clearly not the
23:19case because now we all know that.
23:22The balls can kind of go back up
23:23the hill and you can get just
23:25in the setting like I told you.
23:26If acinar cells they can
23:28become proliferative.
23:29You can get sort of the balls going
23:31over the grooves and being becoming
23:33other cells like beta cells in the
23:35pancreatic islets can become alpha cells.
23:36So these are trans differentiation
23:39and dedifferentiation events.
23:41And in fact when you really think
23:43where we care is pathologists and
23:45and pathology researchers about the
23:47injury and inflammation standpoint.
23:49You know it's quite possible that
23:51none of these grooves even stay the
23:53same during inflammation in the
23:54entire niches changing and all the
23:55groups are changing the identities
23:57may change you know and as we do
23:59more single cell RNA seek we see that
24:01you know I cell identities are all
24:03kind of overlapping you know and and
24:05these groups may not be so so clear.
24:09So there's a lot of interest in
24:12collagenosis and or in itself by in
24:14plasticity and differentiation and in
24:16fact that kind of got I was tickled
24:19to see that there was a last month
24:20the call for in scientific reports
24:22for papers on on plasticity and
24:24specifically specifically pathogenesis.
24:26OK.
24:27So the why do we have this term
24:30pathogenesis and the reason is because
24:32all of those balls are rolling around
24:35on the hill that that I was showing
24:38you had to do sort of with the.
24:40That, that tissue and developmental biology,
24:43the idea that every cell has got its
24:46own identity and that in plasticity
24:48events the cells, you know,
24:49change identity and it matters if
24:51they become less differentiated
24:52than they're rolling up.
24:53And if they're trans differentiated,
24:55they're, you know,
24:56becoming another cell type.
24:57But what if we're actually interested
24:59in the process of how you take a
25:02differentiated cell and convert
25:03it to a proliferating cell?
25:05You know,
25:05that is not likely to be different
25:08in every single organ, just like.
25:10If you need a program cell death,
25:12you have the apoptotic program and you
25:14have apoptosis and that's the same.
25:15And nobody thinks that apoptosis
25:17is different in every cell type.
25:20So this change in identity,
25:22these dedifferentiation events are
25:23likely to be similar across tissue types.
25:26So there must be a cell biological process
25:29or an osis that dictates these events.
25:32And so we came up with this idea that if
25:35we wanted to look at the cell biology
25:38of how these cells rearrange then.
25:40We should have a term
25:41so we can talk about it.
25:42And Paola is the Greek return,
25:44like in palindromes, you know,
25:45a site that goes back and forth.
25:50Can be read both ways right.
25:52And and and Jen is the general,
25:55you know, generative.
25:56So Palingenesis is the return to the
25:58generative state, regenerative state.
26:00So but when we're talking about this then
26:03what we're talking about is basically.
26:05How do you take these chief cells and make
26:07these metaplastic proliferative cells?
26:09So these are very Long live
26:11cells that don't proliferate.
26:12How do they become proliferative?
26:15So the take home is that it?
26:18It's a the.
26:19Basic.
26:20Like so biological change that
26:22has to happen here is a change
26:25in the way the cell uses energy.
26:28When the cell is in the base
26:30of a gastric unit,
26:32then it uses energy to produce
26:34digestive enzymes and secrete.
26:35When it's in the base of a of
26:37a reparative metaplastic unit,
26:39then it uses energy to divide.
26:41So all of the in between.
26:43The Collagenosis part is how the
26:46cell adapts itself to go from
26:48a digestive enzyme secreting
26:50energetic cell to a proliferating.
26:53Non energetic but but non secretory.
26:57So and basically this is the basic
26:59scheme which seems to be conserved across,
27:02you know, from fly guts to, you know,
27:05pancreas to stomach to lung.
27:06Every time you are calling
27:08differentiated cells back into
27:09the cell cycle and that is that
27:12there's a massive upregulation of
27:13autophagy and lysosome as the cell
27:15reprograms its internal organs.
27:17Followed by a second stage where
27:19the genes that we recognize it
27:22as being metaplastic.
27:24And those are a lot of different
27:26genes like trefoil factor or
27:27spasmolytic polypeptide or some
27:29of the socks genes like Sox 9.
27:31Followed by this very important one,
27:33which is the stage when the cell
27:35decides whether to actually
27:36enter the cell cycle or not.
27:38And this is the key stage for cancer
27:40because you're taking these old
27:41long lived cells and you're bringing
27:43them back into the cell cycle.
27:45And so this is a checkpoint that
27:47we'll talk about as being important.
27:48And just to kind of put us on
27:50an ultrastructural footing,
27:51what we're talking about is a very
27:53large pancreatic acinar cell or
27:55digestive enzyme secreting chief cell
27:56with layer after layer of rough ER,
27:58all these secretory granules becoming
28:01this much smaller proliferative
28:03stem like cell.
28:04And this can happen in the mouse and
28:06you know about 42 hours basically.
28:10So the kinds of things that are
28:12going to happen and we're going
28:13to talk about are modeled in this
28:15little video that Jeff Brown is a
28:18gastroenterologist and assistant
28:19professor at Washu now.
28:21Um,
28:21basically all this rough ER turns
28:24into autophagosomes and then starts
28:27to digest all the secretory
28:29apparatus and also gets rid of all
28:32that extra ER itself.
28:33The cell reshapes like
28:35this and then the next step
28:37is that's going to enter the the cell cycle.
28:40So how do we study this?
28:43So what we've taken to do doing is to
28:46looking at these metaplasia models,
28:49both of which involve collagenosis,
28:50both of which are drug induced and
28:53relatively short term like within days
28:55we can get these changes in both the
28:57stomach and the pancreas at the same time.
29:00That way we can look at all
29:01the conserved features,
29:01not just what happens in the stomach.
29:04And so we use two systems for the most part,
29:06one of which.
29:08Juan Jay invented which is our discovery,
29:11which is that if you treat mice
29:13with high doses of tamoxifen,
29:14it has an estrogen and sex independent
29:17toxicity effect on the stomach,
29:19which kills all the parietal cells
29:21within a couple of days basically,
29:23and reprograms the chief cells
29:25and the entire oxyntic mucosa
29:27into this pyloric like mucosa.
29:29And the other is an established
29:31model of of Cerulean,
29:33which is a CCK hormone analog treatment
29:36that turns the pancreas into this.
29:38Kind of duck like,
29:40but it's really just more again
29:43metaplastic proliferative phenotype.
29:45So this is the dosing scheme for
29:47high dose tamoxifen and this is
29:49what it looks like pathologically.
29:50Here's a normal mouse stomach
29:51with parietal cells up here,
29:53digestive enzyme secreting chief
29:54cells here and within three days of
29:57those tamoxifen injections the cells,
29:58the units become like tubes with
30:00just mucus cells on top and
30:02mucous cells in the bottom and
30:04then proliferation throughout,
30:05whereas normally proliferation
30:06is confined to this top area in.
30:09The normal stomach and pancreas,
30:12all of these acinar acini open up
30:14and you get these kind of cuboidal
30:17cyst like proliferative cells also
30:19if we do the cerulean treatment
30:22there just to give him a plug.
30:25To embarrass him a little bit.
30:28So with this system then we've been
30:30able to and I'm just going to show
30:32you some highlights but you know
30:33because a lot of this is published
30:35because it the the stomach system
30:37is so synchronous and then we can
30:40transmit translate that into lesions
30:42in humans and and and then confirm
30:45with the the pancreatic system we've
30:47been really able to kind of pretty
30:50quickly delineate us and others
30:52the the the program that happens in
30:55polygenesis and basically you take an.
30:58A uninjured secretory cell and you
31:00cause some kind of injury that's
31:02going to induce some metaplasia.
31:04And of course you know as we know
31:06the whole point of that is to induce
31:07proliferation so that it repairs the damage.
31:09But the other thing that happens
31:11is about this kind of time course
31:13all the different the organelles
31:14that are specifically tied to the
31:16differentiated function are decreased.
31:18You know,
31:18so things like the rough ER and and
31:20and and this is focused on the stomach,
31:22but they're equivalents in in
31:24pancreas and other organs,
31:25but things like pepsinogen and and so on.
31:29And that occurs across these three stages.
31:32The first stage is this massive autophagy,
31:34which is of course what's helping to
31:36get rid of these differentiated organs.
31:38The second stage is that METAPLASTIC
31:40gene expression where you start to see
31:43that the cells have rearranged how they.
31:45Actually Mark and label and
31:48then the final stage is this.
31:50Mtorc increase, which is critical for
31:53entering into the cell cycle and that is
31:57immediately after a stage of induction
32:00and then suppression of people 53.
32:01So this crossing point is very important
32:03because the main thing that P53 does
32:05is I'll show you is suppress mtorc.
32:07So CP3 has to decrease for these cells to
32:10be licensed to read into the cell cycle.
32:13So you know we're going to head
32:14on this theme several times,
32:15but I already hinted at it from
32:17what we know about Barretts and
32:19why this kind of reprogramming.
32:21Is so important in NYPD.
32:22Three is important.
32:24It's important for this licensing step.
32:26You don't let differentiated cells
32:28back into the cell cycle unless
32:31they've cleared up 53 checkpoint.
32:33So thinking about mtorc one,
32:35it's the central energy regulator and
32:36this is a super simplistic version of it.
32:38But just so that we're on the same page,
32:41you know it's pretty much integrates
32:43the vast majority of the cells
32:45energetic inputs and outputs with
32:47the two main wings being related,
32:49wings being protein translation
32:51and of course driving the cell
32:53cycle via phosphorylation of the
32:55small ribosomal subunit 6.
32:57So this is going to be important
32:59because this is a great marker for
33:01Mturk activity by immunostaining.
33:03Works great or an IF you can tell
33:05how much import there is by how
33:07much phosphorylated S6 there is,
33:09so Amtrak increases.
33:10This in itself is stimulated by
33:12low energy and by autophagy and
33:15all of the breakdown products
33:17in in the lysosomes and a key.
33:19Inhibitor of mtorc is this gene called before
33:22or red one which we'll talk about also.
33:25So let's look at some of how
33:27what this looks like in actual
33:30ultrastructure and you can see
33:31that within 24 hours down now we're
33:34looking in chief cells that we have
33:36all these massive autophagosomes,
33:38auto lysosomes,
33:39all this auto degraded machinery
33:41that these cells start to rearrange
33:43their their entire architecture
33:45and you can see just here this is
33:48quantified by how much lysosomes there.
33:50And then we use this 3D electron
33:53microscopic tactic called focused
33:55IMDb scanning electron microscopy
33:58to kind of look at it more detail.
34:00And you can see as we kind of
34:02spin this around,
34:03this is a single chief cell as this
34:05polygenesis process that's happening early.
34:07This is a capillary loop and
34:09these are the secretory granules,
34:11this is the nucleus and these are
34:12all lysosomes and autophagosomes.
34:13So like half the cell becomes
34:16auto degradative as the.
34:19As this early stage in Polygenesis happens,
34:23so that's what's happening to
34:27autophagosomes and and lysosomes.
34:29For that to happen Mturk has to decrease
34:31and here we're looking at mtorc
34:33activity using this phosphorus 6 and
34:35here we focus on the chief cells.
34:37And here within 12 hours all all of this
34:40phosphorus 6 or M torc activity is lost
34:42in the chief cells and then by maximum
34:44metaplasia it all comes back again.
34:46So here it's working for secretion
34:48and not for proliferation,
34:49here it's working for proliferation.
34:51And in between is when all that autophagy
34:53is happening and you can see even on
34:56Western blots of mouse stomach you can
34:57see it happening on the other hand.
34:59We knock out this suppressive ddit 4,
35:02which I showed you in that cartoon with
35:04it gets induced early to suppress network.
35:07You don't have the same decrease
35:10in mtorc activity and you don't
35:13have the same autophagy.
35:14So if you look at mtorc,
35:15basically it's much, you know.
35:17Normally it's like that and in
35:19the four knockout it's like that.
35:21So that leads to actually more,
35:24more proliferation,
35:25more metaplasia downstream.
35:27And conversely,
35:29when you inhibit mtorc.
35:32That's how we know that the cell cycle
35:35reentry is critical because taking rapamycin,
35:38an M TORC inhibitor,
35:39and treating mice with it does not block
35:42the the metaplasia or the autophagy
35:44or those first couple of steps,
35:46but it it blocks the
35:49proliferation completely.
35:51So early on did it 4 suppresses mtorc.
35:54We have all that autophagy but on
35:56that last slide we also see that
35:58did it four goes away within the
36:00first couple of stages and that's
36:02when 53 comes on and P53 continues
36:04to suppress M torque until or
36:06unless the cell then decides to
36:08come back and the cell cycle.
36:10So that part of the way we know that
36:12is that in P53 knockouts we also don't
36:15have this mtorc loss early on we
36:18have more proliferation both in the.
36:20The stomach and the pancreas and
36:22then what we know that the critical
36:25regulator of P53 that tells the cell
36:28whether the cell should increase
36:30M Turk and go back into the cell
36:32cycle is a protein called ifrd one.
36:34And we'll show you how that works
36:36and how that P3 I 41 access works.
36:39But you can see it's massively
36:42upregulated during collagenosis
36:43and then as the cells we enter
36:45the cell cycle it goes away.
36:47And in the absence of in the
36:49absence of ID 11,
36:50all the cells wind up dying and
36:52not completing the process.
36:54But if you knock out paid 53,
36:56then they're rescued and
36:57they reenter the cell cycle.
36:59So that's how we know I pretty
37:00when it's upstream of 53.
37:01So we'll talk about how RD1
37:03dictates to P3 to dictate M torque.
37:05A lot of this work was done by Max Yao,
37:08who's in China now as an assistant professor.
37:12So let's talk now about some of the
37:15machinery that executes this process
37:17and the way that we've started to do this.
37:20Stepping back,
37:21right,
37:21like if this process of taking
37:23a differentiated cell and bring
37:25it back into the cell cycle is,
37:28you know,
37:29a conserve process across
37:31multiple tissues just
37:32like apoptosis, then there should be
37:34genes that are dedicated to the process,
37:37just as there are genes dedicated to
37:39apoptosis like BCL's and caspases and so on.
37:41So we started doing screens in
37:45these regenerative metaplastic.
37:47Organs after after you know during
37:51the regenerative phase and look for
37:53genes that are all ex Co expressed
37:54and and from these I FD one indeed it
37:56four came out I've already told you
37:58about them need it for suppressing
38:00mtorc I-41 suppressing P53 but we have
38:02other targets that we've been working
38:04on another really strong one is ATF
38:06three which I want to talk about and
38:08we're starting to piece together then
38:09this architecture but this is what
38:11we've learned so far in in this talk
38:13do you injury happens the cell starts
38:15to undergo a topology did it for.
38:18Suppresses mtorc to turn it off
38:19to allow the autophagy to happen.
38:21I have heard one is induced that
38:24eventually accumulates and suppresses
38:25P53 which allows cell cycle entry.
38:30So. Why then is mtorc so important?
38:33Because, you know,
38:34when we think about why Barretts
38:36becomes cancer,
38:37why gastric intestinal metaplasia
38:38or you know,
38:40pseudo pyloric metaplasia gives
38:42rise to cancer and we think about
38:45this pathogenesis process,
38:46this conversion,
38:47you know,
38:47being critical for that and mtorc being
38:50critical for that cell cycle reentry
38:52because that's what you need for cancer.
38:54Why is it so important?
38:55Well,
38:56here's what we delve like the deepest
38:57into the structure and organelles
38:59before we kind of come back out again.
39:01Our thinking now is that it's all about
39:05ribosomes when you're a chief cell.
39:06I showed you that electron microscope
39:09micrograph where it's just layer after
39:11layer after layer of rough ER and all
39:14that roughly RISER line by ribosomes.
39:17That are making digestive enzymes to go into,
39:20you know,
39:20the lumen of the R and then to be secreted.
39:23When you become a proliferative cell,
39:25you don't need all that secretory
39:26roufi R you need ribosomes in the
39:28cytosol to make more ribosomes than
39:30histones to make a copy of the cell.
39:32And the key driver for ribosome
39:35Biogenesis is M torque OK,
39:38and the reason why ribosome Biogenesis
39:40needs so much energy is because it's
39:43an incredibly complex process of
39:45assembling all of these ribosomal
39:47proteins and ribosomal RNA's that
39:49require all three RNA polymerases
39:52and translation into these.
39:53Large and small 1640 subunits which
39:57come together as a single subunit,
40:00multiple modifications happen and all
40:01of its sort of starts in the nucleolus.
40:03So that's our basic ribosome review.
40:06And then as I talked about,
40:07there's a big difference between this
40:09pool and this side of solid pool,
40:11right,
40:11because this is for secretion and this
40:13is more for division and housekeeping.
40:16So to get from the ribosome to translation,
40:20we have to realize that the M
40:22RNA is going to be loaded up
40:24the preinitiation complexes.
40:25That's going to bring the two
40:27subunits together.
40:27So the two subunits only
40:29come together with M RNA
40:30normally, OK. So they're kept together
40:33with M RNA as they translate.
40:35And then the way most of our
40:37translation happens is not with
40:38single ribosomes but multiple ones
40:40like pearls on a string, line up,
40:42line up and those are called polysomes.
40:44We're not going to go too much into this,
40:45but you can tell the difference.
40:47Between Monisms and polysomes by
40:48spinning them down and the longer
40:50you know ones or polysomes so they
40:52take lower longer to spin spin out.
40:54So the last review slide here on ribosomes.
40:57The reason why they require so much,
40:59they require 80% of the cells energy.
41:02So that's why it's so important how you,
41:03you know, regulate ribosome Biogenesis and
41:0660% of your RNA in each cell is ribosomes.
41:10So there's huge proportions of the
41:12transcription and translation that
41:14goes into ribosome Biogenesis.
41:15So what happens to ribosomes
41:17during palingenesis?
41:18So we knew already that they had to
41:20be coming off the rough ER and we saw
41:23that's what all the autophagy was doing.
41:25But you can also just document it,
41:26there's many ways.
41:27To to show that you're losing
41:29both large and small.
41:31Subunits of ribosomes are just Western
41:33blots early on in the process and
41:35then they come back on again later.
41:37So there's a loss and then
41:39regeneration process.
41:39But you can also see some of the
41:42ribosomes getting taken up into
41:44the the rough ER and you can also
41:46see them kind of spinning off the
41:47ER here into the sideshow.
41:49And in fact Juan J was one of the first
41:51to show this by knocking out a gene
41:53that that regulates all that rough
41:55ER when he was a graduate student.
41:57So this is kind of what we think
41:58is happening.
41:59In terms of stages of of pathogenesis,
42:02normally you have all these rough ER.
42:06Ribosomes making peptides and then,
42:08you know,
42:09there's an injury and these autophagosomes
42:11start to take up the raffia and the
42:13ribosomes all come off OK what's the
42:15problem with the ribosomes coming off?
42:16As soon as they come off the M RNA,
42:17then they fall apart into their
42:20subunits and into ribosomal proteins,
42:22and those can stimulate P53.
42:24I'm going to show you that again
42:25a couple of different times,
42:26but that's probably why this whole ribosome
42:29is the center of this mtorc P53 axis.
42:31But this is just to show you that we
42:33also get a lot of ribosome Biogenesis,
42:35so we're losing.
42:36Have some and then later we see
42:38huge increases in nucleolar size,
42:40which you can see here in quantify
42:42in both the stomach and the pancreas.
42:44So what that means is we're losing
42:46ribosomes here and then the nucleoli
42:48are getting turned on or making
42:50more ribosomes here.
42:51But that's not the entire story as we see,
42:54because I 41's going to play an important
42:56part in between those two things.
42:58So to be able to study these things,
43:00we already have one tool which
43:02is the ID one
43:03knockout. But Charles Chow in the lab,
43:06who's an instructor looking for a job soon,
43:09also made a knockout of ribosome
43:11Biogenesis for the first time,
43:12surprisingly that he can.
43:14Reduced ribosome Biogenesis knockout by
43:16knocking out this key modifier that's
43:18critical for the small subunit of ribosomes.
43:20And when he does that you that you
43:22can no longer make ribosomes and
43:24when you do that and you induce
43:26collagenosis all the cells die unless
43:28you also put them on a P53 knockout.
43:31So again PD3 knockout is critical
43:33that's sensing the death of of cells
43:37that don't make ribosomes anymore.
43:39So this particular gene which is
43:41involved in the ribosome Biogenesis.
43:44Suppresses P53 presumably because
43:46it makes both subunits.
43:47So they're both subunits are there.
43:49It stops the people to three
43:50induction that happens with
43:52ribosome will breakdown products,
43:53but I have 41 is occurring here
43:56earlier I showed you and it's also
43:59responsible for suppressing P53.
44:00How does that work?
44:02Well, it turns out that it's in between.
44:04That's just to remind you of
44:05that and that NAP 10 is there,
44:07but I heard you once turning on
44:08earlier and doing the suppression.
44:09So how does it work?
44:12So it turns on it, you know,
44:13it turns on here.
44:14And what it does,
44:16it turns out.
44:17Is that I 41 fits right here right where the
44:20M RNA would go between the two subunits.
44:23So when I offered you one
44:24attaches just like M RNA,
44:26it can keep the two ribosomal
44:27subunits together as a whole.
44:29So instead of having this happen
44:31during those early stages,
44:32which then leads to breakdown
44:34in P53 activation,
44:35I 41 can come right there in that pocket.
44:40And as they come off the ribosomes,
44:41they're preserved.
44:42So essentially 53 is blocked because
44:44you don't get breakdown of all the
44:47ribosomes during the first stage.
44:49So on the one hand you could have this,
44:52but when you have ribosome Biogenesis
44:54you can stop P53 by making new ribosomes,
44:57and if you have 41 then you salvage
44:59the existing ribosomes so both
45:01of those then converge on P53.
45:03OK, so that is the ********.
45:06Organellar and molecular stuff.
45:08So now let's come kind of back out to
45:10how this all comes out in tumors and
45:12and come back out towards the pathology.
45:15So with all this background
45:16then it's pretty clear,
45:18you know that the cells spent a lot
45:20of time trying to regulate them to
45:22work via PD3 and via this protein deed
45:24at 4:00 to be able to ensure that
45:26the there's no tumors that come out
45:28of this taking these old cells and
45:30driving them back into the cell cycle.
45:32So what if we get rid of the ability to
45:35stop mtorc and regulate this process so.
45:38You know what if we take out them
45:40torque regulation and then in
45:42in a system where we can induce
45:43metaplasia multiple times and the
45:45thinking would be then that what's
45:47going to happen is we kind of
45:49injure each time and we don't
45:51have much error checking.
45:52Then you go through collagenosis,
45:54then you heal, then you go through
45:55pathogenesis and you heal.
45:56But each time you can accumulate
45:58mutations until finally you get to
46:00the mutations like Karas or something
46:02like that that drives a tumor and
46:04then you know you no longer go
46:06back to being a chief seller and.
46:08Lesson or so.
46:09So I already showed you how we kind
46:11of we do these screens and coming
46:13back to dead at 4 so that you know
46:16knocks out the ability of the cell
46:17to decrease M torque and it knocks
46:19out its ability to be able to
46:21sense the P53 damage and to be able
46:23to stop cells from coming back.
46:25And cell cycle basically just kind of
46:27skips past all this error checking
46:29right into the proliferation.
46:30So you see a lot more proliferation
46:32when you knock out deed it for.
46:33And So what happens is essentially
46:35you can take mutations and carry
46:37them right into these dysplasias.
46:38And so functionally what Max did
46:40in the lab was do multiple rounds
46:43of Immunogen which causes gastric
46:45tumors kind of slowly in the stomach
46:48in these cells that could no longer
46:50in these mice that could no longer
46:52regulate the the collagenosis and
46:54that mtor checkpoint versus control
46:56cells that could still did multiple
46:58rounds of tamoxifen to do multiple
47:00rounds of metaplasia and repair.
47:01And what he saw as we predicted was
47:03a lot more tumors in the deed at 4
47:06knockouts and a lot bigger tumors
47:08in fact just for the pathology.
47:10This is one that arose as a huge
47:12sort of polypoid tumor that was
47:14more intestinal type between the
47:16Antrim and the corpus,
47:17but then had a had a focus of the diffuse
47:21signet ring cells that you can see.
47:23And it's rare in the mouse to get
47:25such an obviously metastatic tumor.
47:26You can see them kind of in this
47:28PAS stain going right through the
47:30muscle area and into this aerosan
47:32intelink vascular space.
47:34So in other words, if you can't do this,
47:36check here to make sure these
47:38cells are OK and send them back,
47:40you know, to repair them.
47:41They come back to repair with mutations
47:44and then eventually they form tumors.
47:46OK, so last thing, the human thing.
47:50You coming back all the way back to human,
47:53the human part of the talk.
47:56Again,
47:56we've been again going back to
47:59Barretts and trying to study this,
48:01how these processes happen and how
48:03people heal from these processes.
48:05Unfortunately,
48:05this great mouse models that we
48:07can use for tumorigenesis and
48:09metaplasia and stomach don't apply
48:11because mice don't get variants,
48:13they don't reflux at all,
48:14they don't have any bile or
48:15acid ever in their esophagus.
48:17So there's no really good rodent
48:19models for this.
48:20So you know, you have to study the human.
48:22And so I've been collaborating in
48:24this amazing collaboration with.
48:26Rhonda Souza and Stu Spechler's
48:28group and Rob odds.
48:30Also, you know, pathologist,
48:31yeah, pathologist.
48:32To look at, at their models of
48:34Barretts and some clinical trials,
48:36I'll just show you.
48:37Here's where I had them down to Houston.
48:38There's Rob and and me.
48:41And there's actually, there's my.
48:42There's the same microscope that Wanj
48:45learned on, taken down to down to Houston.
48:48And Rhonda like is fond of saying
48:50that humans are the best model system.
48:52So it with Barretts we have to do that.
48:54So, So what in this model what
48:57they've done is they you know with
49:00the dysplastic Barretts you can treat
49:02it by radiofrequency ablation just to
49:04basically take out all the Barretts
49:06and take it down to the ulcer bed and
49:09granulation tissue and then for some
49:11reason it heals back as squamous.
49:13So basically what you're doing
49:14is radiofrequency ablation,
49:15the Barretts,
49:16and it goes to this just ulcer bed basically.
49:18What's leftover?
49:21And then you know what happens though,
49:23you know after this ulceration is it
49:25comes back as a squamous and what
49:27they did was they they took a bunch
49:28of patients and enrolled them should
49:30also say for the this study and
49:32then did the pre and then one week,
49:34two week and four week biopsies
49:35all the way proximal to distal
49:37from before the margin of RFA to
49:39the gastric margin after the RFA.
49:41And you know try to look at how
49:42the healing process, how all this,
49:45you know mucosa became squamous again.
49:49So that's kind of what it looks like.
49:52You know, the question is where
49:53does all that squamous come from?
49:54And the only source of squamous or even
49:56epithelial cells that you could think of
49:57would be at this proximal margin, right.
49:59But it turns out that's
50:00actually not what happens.
50:01What happens is it comes
50:03back as squamous throughout.
50:05So there's some source of squamous
50:07epithelium that's obviously trans or D or
50:10some kind of differentiating, you know,
50:12that's that's feeding the squamous.
50:14And you know we have a couple of clues,
50:17one of which well we talked about.
50:20But one of which I'll show you
50:22evidence for here, you know,
50:23so the idea is that coming from
50:24the proximal squamous, you know,
50:25there's a come from the distal gastric,
50:27but then why would that be squamous?
50:28But it turns out it just comes in
50:30all these little islands like this.
50:31And so if you focus,
50:32here's one of these islands of this
50:35NEO squamous healing epithelium.
50:37And, you know,
50:38where does this come from on either side?
50:40Basically it's going to go down
50:41to like a single cell.
50:43It turns out that there's pretty good
50:45evidence both morphologically and also
50:47with their advanced endoscopy that
50:48if you look under each of these new.
50:51Kind of ulcerated surface as a single
50:53cell layer of squamous is forming.
50:55They're all underneath ducts
50:57from submucosal glands.
50:59So you know,
50:59just for those of you don't remember
51:02your human esophageal theology.
51:04These are the 70 coastal glands,
51:05and they have ducts that reach
51:07up to the squamous epithelium.
51:09And normally like if you blade
51:10all this with RFA,
51:11then there's still a source of epithelium.
51:13At least you know distally for
51:15some of these squamous islands.
51:18But the other source is probably some of
51:19these deeper Barretts that escapes the.
51:21RFA as we have a lot of work showing
51:23that there's transitions in there.
51:26So like in fact that's what we're doing now.
51:27We're doing spatial transcriptomics,
51:29we're growing organoids and we're doing
51:31a lot of IHC and Multiplex IFF to kind
51:33of show how these transitions happen.
51:36So that's that. So summarizing.
51:39Take homes.
51:41This kind of pyloric metaplasia
51:43is some kind of like maybe
51:45or metaplasia that you see,
51:47you know,
51:47intestine in the cases of SL going
51:50towards that you see the body of.
51:52I don't mean gastritis and H
51:55pylori induced atrophic gastritis,
51:57it's the what seems to be
52:00happening in Barretts.
52:02And the root of this and although we
52:04don't know this yet in the SL how
52:07that happens but but at least in the
52:09pancreas and the stomach for sure
52:11and probably in Barretts is the cell
52:13biological process that's driving this,
52:15the palingenesis process.
52:16And that basically is about cells converting
52:19energy from one state to the other.
52:21Now you know this is a pathology grand
52:24rounds and I'll tell you that when
52:26I was doing a lot of this looking
52:28at where this metaplasia happened
52:30and where what people thought about
52:32it 100 years ago.
52:34Well, over 100 years ago,
52:35George Adami was a famous pathologist
52:37who at the time was at McGill said,
52:39you know, it looks like in tissues
52:41that are going to become cancerous,
52:43there's all this reprogram,
52:45you didn't use that term of cells
52:48from mature cells to dividing cells,
52:51and that seems to fuel the cancer.
52:52So he kind of anticipated all of this.
52:54And he said that what must happen
52:56is the cell converts its energy
52:58use from secretion to division.
53:00So, you know, it's kind of funny.
53:02Then we forgot that for like 9000.
53:03Years.
53:04And then, you know,
53:05we've come back to that old
53:06pathologists who just by looking
53:08at a bunch of tissues made the
53:10same kind of analysis that it
53:11was the same in multiple tissues,
53:13you know,
53:14even as a picture of a liver cell with
53:16its kind of autophagy before they
53:17even knew what the organelles were.
53:19So a lot of that depends on ribosomes
53:21and and so the metaplasia depends on
53:23this collagenosis which depends on ribosome.
53:26So these are all areas where you
53:28could target potentially both to.
53:30First metaplasia,
53:31but also if cancers emerge from
53:34those this aberrant checking
53:36of pathogenesis or P53,
53:38then maybe with they proliferate
53:39by going through that.
53:48The city. Ohh, it's all the eye.
53:53And where we got some of the
53:55mice and this is our group down
53:58in Texas with my wife's lab,
54:00she's mysorekar and on the mills,
54:02so we're the M&amp;M labs together, so.
54:11Yes, you know. And then.
54:14So, so I don't know,
54:16but I think there are papers already too.
54:19But I'm, I I bet you it's the same aisle 13,
54:21aisle 33 access which drives it
54:24seemingly in in Barretts and
54:27in pancreas and and in stomach.
54:30The, the very idea Polygenist
54:33is absolutely reversible.
54:34Yeah, 100% it's,
54:35it's a normal way to recruit stem cells,
54:38especially for organs that don't
54:39have stem cells like the pancreas.
54:41That's the only way the pancreas
54:42can kind of repair itself is by
54:44recruiting the acinar cells.
54:45And then normally they come right back.
54:47It's only when you know they acquire
54:49enough mutations that they don't read,
54:51differentiate and they think
54:52it's an idea to keep growing.
54:53You know, that it becomes irreversible.
54:55And that's why we think,
54:57you know, chronic inflammation,
54:58which is the first question you had,
55:00is so important.
55:01Because it keeps stimulating
55:03this collagenosis until of these
55:05kind of old cells.
55:06You know, if you think about it,
55:07they don't really do much error
55:09checking of their chromatin under
55:10normal circumstances because
55:11they're just making a handful of,
55:13you know,
55:14digestive enzymes over and over again.
55:15And most of their ribosomes
55:17are already taken care of,
55:18so they're most of their chromatin is inert.
55:20So then you ask them to rearrange everything,
55:22come back into cell cycle and
55:23expose a bunch of cell cycle genes,
55:25which is very dangerous.
55:26So they need this error checking and it
55:29just seems like we've evolved only one.
55:30Protein which is P53 to do all
55:32that error checking.
55:33So each time you go through that cycle of
55:36you're asking people to three to work.
55:39And the more you do it the more chances
55:40you're taking until you get a you
55:42know clone that doesn't have it work.
55:43And then you start having more
55:46errors in each replication.
55:47And then when that happens then
55:49eventually you'll get a make or
55:51a rass or you know something else
55:52that drives it outside the geotrack.
55:54Yeah.
55:55Actually you know I 41 is conserved
55:57all the way through plants.
55:58It's the the the the it's.
56:01Amazing protein.
56:01It goes right between the ribosomes.
56:03That's why it's so conserved.
56:04And it has 0 phenotype in any Organism,
56:07from plants to flies to yeast.
56:12Even if it's not in all yeast.
56:13Because I think it's more multicellular
56:15thing when you knock it out until
56:17you injure and ask them to kind of
56:20reprogram and respond to injury.
56:21So there's flying effort you want and
56:23if you knock it out then you can't
56:25recruit stem cells and the fly gut.
56:29Deliver after partial hepatectomy
56:31of you knockout I31 you screw up
56:34the ability to to get all that GI
56:37tract again and parasites kidney.
56:40That's a non GI Oregon also and in
56:42fact all this is tied to aging in
56:44the sense that as you get older
56:45you seem to be able to lose.
56:47You lose these markers in these
56:48genes and in in the bladder we
56:50know that actually where each time
56:52you go through UTI of shedding you
56:53need to recruit new stem cells.
56:55As you age you lose I 41 and
56:59you're less able to do this.
57:00That's work from the My wife side actually
57:03because she's a a bladder expert.
57:15Yeah, right. So, yeah,
57:16the question is why are some metaplasia
57:18is dangerous and some not, right?
57:20You know, I have no idea because that's
57:22the same thing with stomach, right?
57:25I mean, autoimmune gastritis
57:26causes massive metaplasia.
57:27And you know, there's a huge controversy
57:30about whether it increases risk of
57:32gastric cancer or not in the absence
57:34of Co infection with H pylori.
57:35And I think probably the
57:37consensus is it doesn't.
57:38So even the very same metaplasia
57:40and H pylori context.
57:42You know it's risky, but but it's not in
57:44the autoimmune gastritis context, so.
57:49I, I, I don't know, uh, I, you know,
57:52I think 1 aspect would be the
57:54repetitive nature and the chronicity.
57:57Another aspect, you know,
57:58in the stomach,
58:00I've always thought of that H pylori is
58:01also got oncogenes that it, you know,
58:03pretty much injects into cells.
58:05And also there's this sense of kind
58:09of progression and that that that
58:12that glands on the border between
58:14the Antrim and the corpus going to go
58:16through this more and more and more
58:17as autoimmune gastritis, I think.
58:19You know,
58:20kind of happens sporadically,
58:21hits an area,
58:22then comes back and it's kind of back and
58:24forth in different areas as opposed to
58:25the same area going over and over again,
58:27but.
58:29I've never been asked that question.
58:30It's a really good about the cervix,
58:32you know like in areas where you
58:34get metaplasia that don't that
58:36may even be protective.
58:37I mean you know in the stomach a
58:39complete intestinal metaplasia seems
58:40almost protective against gastric cancer.
58:42So that's another interesting fact.
58:46And and I think in autoimmune
58:48gastritis there's more complete
58:50than there is incomplete.
58:51But I think it's definitely risky
58:53to have the kind of metaplasia where
58:55you have a mixed phenotype where it's
58:57both gastric and intestinal and it keeps.
59:00Happening it almost, you know,
59:02is asking for trouble.
59:03So maybe pure metaplasia are better.
59:05I don't know.
59:06It's a good question.
59:08Haven't.
59:11Haven't asked.
59:13OK. Question on the.
59:18Building. And you describe.
59:23But when you look at.
59:26Or the before.
59:31Yeah, yeah. He.
59:37Your life experiences that are known
59:40to alters. So the question is, drew,
59:43are there germline variants of genes
59:45like D at 4 the AG is the autophagy
59:49genes that affect susceptibility?
59:51I. That's a good question.
59:54I don't know did it.
59:55Four is very controversial also
59:57from the cancer standpoint,
59:58it seems like half the literature
60:00says that mutations are variants or
01:00:02pro tumorigenic and half are anti.
01:00:04But the issue with pathogenesis
01:00:06and tumorigenesis is you know it's
01:00:09a cycle normally so umm and it's
01:00:11sort of aberration in the cycling
01:00:12that we think is giving the tumors.
01:00:14So just kind of completely knocking
01:00:17it down might not would probably
01:00:19give you a premature aging thing
01:00:20if in fact that's what I said,
01:00:22I pretty one has no phenotype
01:00:23but actually it has an aging.
01:00:24Genotype so as you age then and
01:00:28you get inability to regenerate
01:00:30the that tends to be where you
01:00:33manifest your pathogenesis defects
01:00:35because you probably wouldn't be
01:00:37able to necessarily you've never
01:00:39traced people that don't get tumors
01:00:41based on you know lacking that but
01:00:43obviously people do three is a key
01:00:46checkpoint and that is the you know
01:00:48incredibly tight the tumor genesis
01:00:50the in terms I'll be a little bit
01:00:53more specific though about autophagy.
01:00:55Which is that we have tried with a
01:00:58G57 and 1601 variant to show effects
01:01:01and haven't really been successful.
01:01:04Where we have genetically been able
01:01:06to completely shut palingenesis
01:01:07down both in the pancreas and the
01:01:09stomach is by affecting lysosomes.
01:01:10So if you want to really get dive
01:01:12into the autophagy aspect of it,
01:01:13we actually think it.
01:01:14It's from the EPG 5 which is the
01:01:16fusion of autophagosomes and
01:01:18lysosome steps down there are the
01:01:19most important and a lot of it
01:01:21maybe non canonical autophagy.
01:01:22So a knockout the the best knockout.
01:01:25They had to stop.
01:01:26The whole process is as in the
01:01:29phosphorylation that phosphorylase that puts
01:01:31phosphate phosphate groups on Mano six,
01:01:34you know to make Manor 6 phosphate.
01:01:36So none of the digestive,
01:01:37the license only enzymes go to the lysosome.
01:01:39Those mice are completely resistant
01:01:41to you know which is not necessarily
01:01:43a good thing because it means
01:01:45they can't repair in the pancreas
01:01:46is kind of if you keep forcing
01:01:48pancreatitis or pancreas is turned
01:01:50to snot basically because they can't
01:01:53you know repair the damage so.
01:01:55In our experience,
01:01:56it's really lysosomes I you know,
01:01:58it's massive autophagy.
01:01:59Clearly LC3,
01:02:00it's all the classic but the the main.
01:02:03The thing seems to be required is
01:02:05the flux through the lysosomes.
01:02:09Short question.
01:02:12Cheap.
01:02:18Yeah. So, so the question is whether
01:02:20parietal cells can do the same thing.
01:02:22And in fact, as part of the more general
01:02:24question of is it like universal and
01:02:26the parietal cells are great test case
01:02:27of the only cell that we've never seen
01:02:30couldn't do any kind of plasticity.
01:02:32And actually Juan Jay also did
01:02:34the that the experiment early on.
01:02:35So if he did when he was doing
01:02:37the the tamoxifen to be marked,
01:02:40all the parietal cells,
01:02:41they all died basically and they they didn't,
01:02:45they never seem to. D differentiate.
01:02:48Actually we have a pretty good idea
01:02:49because some of our work is just on
01:02:51the regular differentiation parietal
01:02:52cells and there seems to be a
01:02:54checkpoint and their differentiation,
01:02:55after which they are no longer
01:02:57plastic at all, but up to about
01:02:59halfway into becoming a parietal,
01:03:01so then they can take detours.
01:03:03And in fact,
01:03:05working with Shilpa Jane at Baylor,
01:03:08we've been collecting some of the
01:03:09interesting sort of parietal hyperplasia
01:03:11that happen in a neuroendocrine setting
01:03:12or an autoimmune gastritis setting,
01:03:14and you can definitely see some pretty
01:03:16odd using markers that we know of.
01:03:18Pre parietal cells some odd
01:03:19sort of parietal cell variance,
01:03:21but I don't think those are coming backwards.
01:03:23I think those are actually coming
01:03:25from the stem cell and then in
01:03:27the setting bottom you gastritis.
01:03:28They take a detour because they're
01:03:30going to be destroyed basically by
01:03:32the anti parietal cell antibodies.
01:03:34So yeah, not all cells can do it.
01:03:36Seems like protocells are quite resistant.
01:03:40A lot of questions.
01:03:45Yeah.
01:03:51He said great. It's great to like,
01:03:54you know, don't present that often,
01:03:56but before a bunch of pathologists, so.
01:04:01Yeah, is the neuroendocrine proliferation,
01:04:04you know those little tumors or little
01:04:06growths or you know that you get with
01:04:08chronic bridal cell loss or chronic,
01:04:10you know, PPI's and.
01:04:11You know, is are those metaplastic?
01:04:13They sure look funny, right?
01:04:15I mean, you know,
01:04:16they don't look like they're normal
01:04:19endocrine cells sitting lining up
01:04:21with the rest of the epithelium,
01:04:23because normally integrins
01:04:25cells are always surrounded by.
01:04:28Other non neuroendocrine epithelial
01:04:29cells and then you know in these
01:04:31lesions you get these little,
01:04:33you know, expansions.
01:04:36And I yeah great great question.
01:04:40How would they you know the the
01:04:41only the one thing that that might
01:04:43speak to that is one of the detours
01:04:46it seems like those riddles can
01:04:48make but I just said is towards
01:04:50more of an endocrine lineage.
01:04:51So you know maybe maybe that's why
01:04:53I never really thought about it.
01:04:56We had a mouse model where we drove
01:04:58large tea energen you know to drive
01:05:00proliferation and to to try to get
01:05:02a bunch of pre parietal cells but
01:05:03what happened with time this is
01:05:05when I was in Jeff Gordon's lab.
01:05:06What happened with time was they all
01:05:09turned into endocrine tumors in the stomach.
01:05:11So they actually went through.
01:05:13So it's like they hit a certain wall
01:05:15of parietal cell differentiation and
01:05:17then took a detour towards endocrine.
01:05:20Then it became endocrine proliferations,
01:05:21and then they became metastatic
01:05:24neuroendocrine tumors.
01:05:24So I don't know,
01:05:26maybe we just solved a mystery.
01:05:28Maybe it's because the reason why they
01:05:30happen so much is not just because of
01:05:32hypergastrinemia and the G cell stimulation,
01:05:35but also because.
01:05:36The pre parietal cells themselves can
01:05:39fuel endocrine cells in that setting.
01:05:42Yeah, and take a detour.
01:05:44They clearly can in the mouse we showed.
01:05:46That means it's hard,
01:05:47it's artificial because we're
01:05:48expressing large T but but still,
01:05:50they start off as pre parietal cells and
01:05:52then you could watch them even become,
01:05:53you know,
01:05:54through EM and and staining become endocrine.
01:05:57So yeah,
01:05:57maybe,
01:05:58maybe that's maybe your two
01:06:00questions are linked.
01:06:06All right. Thank you, everybody. Yeah.