Today I've been asked to speak about our work in this insulin or IGF receptor signaling pathway. This is a pathway we kind of stumbled upon in our work and I hope to convince you that this is important for breast cancer and progesterone receptors can integrate signaling pathways including the insulin receptor alpha or fetal insulin receptor pathway. They can drive a non- proliferating or sort of G0 cancer hallmarks such as stemness biology and phosphorylated receptors can serve as biomarkers for cancer spread. Disclosures are here. So you know we know that one in eight women will develop breast cancer and we have things we can control and things we can't that feed into breast cancer risk, age, genetics, breast density, hormone exposure, and reproductive history for example and things we can control include diet and exercise and lifestyle in some situations and even life circumstances and we know that these things can all interact and factor into complex relationships that regulate risk. So in the context of obesity which is this session we know that white adipose tissue is an active endocrine organ and obesity associated metabolic changes occur. There's increased insulin and insulin signaling and we'll get back to this a little later the mTORC1 and 2 pathways are elevated. There are pro-inflammatory cytokines and growth factors and it creates a milieu within the breast tissue microenvironment that is highly secretory and we see increased hormone synthesis and conversion into cytokines and growth factors and this adds up to greatly increased risk of breast cancer with metastatic behavior. This can be found in the literature by just taking a quick look about this. It's really important and it makes sense for the endocrine society to host a session like this. My lab doesn't really focus too much on obesity but I think this biochemical and signaling story is relevant to that. So in breast cancer we have the expression of the related steroid hormone receptors and today's talk will focus primarily on the progesterone receptor. So we look at progesterone receptor expression in breast cancer as a marker that estrogen receptors are activated because it is a progesterone receptors are an estrogen target gene and PGR expression is estrogen induced typically. And my lab is kind of focused on this hypothesis over the years. We believe that in the context of early oncogenic events where kinase pathways come on that steroid receptor action is greatly altered and this leads to altered hormone responsiveness, altered transcriptional activity and stability of the receptors, and altered promoter selection and an altered transcriptome. And these of course lead to altered cellular behaviors. And today I'll be focusing a little bit more on the cytoplasmic role of these receptors and how they can form signaling nodes that feed into altered biology. And so here's your classical picture of how steroid receptors act where they can bind ligand and come into the nucleus and they do this in a rapid and dynamic manner where they shuttle in and out of the cytoplasm in the nucleus to regulate genes in this classical manner. It's been understood for many years that these receptors can also form complexes that can activate SART kinase directly and this is very much an underappreciated role of these receptors. They can activate MAP kinases in AKT within minutes or even seconds and this role can regulate genes independently of their transcriptional activity. We now understand that these interactions between ER and PR that occur at the membrane and were first described at the membrane can also act in the nucleus. And so now we understand that this crosstalk is very important. It changes hormone sensitivity and responses to therapy. And another way that these receptors rapidly action and rapidly signal is shown here where in my lab in collaboration with Julie Ostrander and Ganesh Raj at UT Southwestern we showed that ER and PR can form complexes in the cytoplasm with this molecule known as PELP1. And the function of this complex seems to be to recruit SRC3, the co-activator, and get it phosphorylated. That complex can also be found in the nucleus regulating genes and we think that it's a mechanism for promoter selection. We also identified a similar complex in triple negative breast cancer with the glucocorticoid receptor and HIF1 and 2 and found that these were stress induced and could regulate the aryl hydrocarbon receptor. I won't talk too much about that today but I'll be coming back to this PELP signal relevant to the to the insulin signaling story. Okay so we were one of the first labs to publish the ER-PR crosstalk and that it's constitutive in breast cancers. These are eight tumors or nine tumors that were found in our clinic and we pulled down PELP1 and could see PRB and ER-alpha constitutively interacting with PELP1 in these tumors. This was performed by Ganesh Raj, our collaborator, and then we defined this complex shown both in the cytoplasm and the nucleus. We then showed in unpublished work that this complex forms under the influence of phosphorylation events and so when we mutate this key serine residue on the progesterone receptor the complex is less stable and we don't see the ER-PR interaction as well when we mutate that serine as a map kinase site on the PR. We think that that interaction requires this phosphorylation event and so now we have this sort of improved picture we think of ER and PR as as playing at the membrane a little bit being highly responsive to kinases and we defined numerous of these interactions through biochemical work over the years and we showed that these kinases map kinase CK2 and AKT could collaborate and phosphorylate the ER-PR complex on both ER and PR and that this complex then very stably can enter the nucleus and regulate genes and it cooperates greatly with IRS-1 and I'll show you that today. So we can now place IRS-1 in this complex. So this is the insulin signaling pathway and it shows you that insulin receptors and how they can coordinate to both the AKT and the map kinase pathway and we found this so quite some time ago in an early gene array where we were looking at ligand independent genes. We're quite interested in how steroid receptors can regulate genes without their cognate ligands and so we set up a screen to look for progesterone receptor regulated genes that didn't necessarily require progesterone and we came up with this insulin-like growth factor receptor signaling pathway and namely IRS-1 was quite elevated and it required the phosphorylated progesterone receptor but not progesterone and this is kind of an example of how these genes that do this behave. We were confused by this at first because if we made a phosphomimic model for example this is a model which is heavily phosphorylated or de-sumylated. You can see very high expression of IRS-1 mRNA and then when we mutate that serine this would be a heavily simulated receptor. We don't see it but oddly we see a decrease in both progestin and antiprogestin and so we were like wow what's going on here what could this mean and we thought well maybe this is really an estrogen regulated gene that requires the presence of PR and so we set up a screen to treat cells with estrogen now and either have them lack PR or have PR present and we found that in the presence of PR without ligand with estrogen treated we found about 40 genes that required the presence of PR and so we thought this might be a scaffolding action of the progesterone receptor in the estrogen receptor complex and we validated some of these genes here. You can see that there were some PR dependent genes that were up by estrogen and in the TCGA they're up in invasive breast cancer. There were down regulated genes also that we treated with estrogen but required PRB to be down regulated and they were also down in the TCGA in invasive breast cancer and we validated some 38 genes that were estrogen induced that required PRB and showed that if you had this signature you did really badly on endocrine therapy and so this is public data now over a thousand tumors where just this little gene signature of 38 genes that are in this scaffolding complex with phospho PRB and ER can really dramatically change outcome with endocrine therapy and these genes validate very nicely. Here's an example of five genes that are up with estrogen but require the presence of PRB. The PRA isoform doesn't do this. It's only the PRB and that PRB is what we find in the complex with ER alpha and so we had this hypothesis that you know we need ER to get PR expression. PR expression goes up we have PR. We can sometimes see inhibition of ER actions with progesterone and that's certainly been reported by Tilly and Carol group in a really nice paper they had but we think that this requires sumo. It's been reported by Kate Horowitz that the trans repression of ER by PR requires this sumo modification on PR and that will actively repress ER. However when we have protein kinases involved in cancer that context changes very dramatically. We get phosphorylation events numerous of them that eliminates the simulation event and now we have a trans activating complex which will activate genes together with ER and PR and drive a different biology not proliferation but actually stemness and metastasis and that's the hypothesis that we've been working toward. Here's another way to show it here where we have repressed genes that have ER and PR and these bring IGFR to them. I'll show you that in a minute and then under the influence of high MAP kinase in the context of cancer there are phosphorylation events the IGFR will disappear and the IRS one will come into the complex and here's some data following that will show you that so we made an antibody to phosphorylated PR and just tried to see what were these target genes and how was this acting in cancer and here's just a picture of the phospho PR and a tissue. We then looked at a tumor tissue microarray and we can see a little over half of the ER positive luminal tumors have phosphorylated progesterone receptors and when we took that phospho PR target gene signature on the left we could see that now only 15 genes confer poor outcome on endocrine therapy and this is a PDX model now where we see interestingly using a total monoclonal antibody you just don't see very much PR it's not very impressive in this PDX model and then surprisingly when we use our polyclonal antibody to the phospho PR we now see a really good signal in these primary tumors that are PDX models and really interestingly their metastasis now in this model is loaded with phospho PR that's nuclear and focal then we did single cell RNA-seq on these metastases and we saw the elevation of PR and PR target genes KLF4 and IRS-1 just like we saw in vitro and we saw the loss of the insulin-like growth factor receptor as has been reported in the literature for endocrine-resistant breast cancer and so these changes recapitulated what's been published in the literature and what we see in vitro very nicely in the PDX model so then we took a closer look at this crosstalk with IRS-1 and we thought well what if IRS-1 and PR are interacting and here you can see really nicely with estrogen we see the interaction by proximity ligand assay of both IRS-1 and PR and so they're coming together in the nucleus if we treat with a progestin synthetic progestin R5020 that the spots are in the cytoplasm and we see this interaction then with IRS-1 if we mutate that serine that map kinase site that keeps the ER and the PR together we lose that interaction and so that interaction requires that phospho site brings in IRS-1 to the complex and we ask well what is it doing so you can now see PR on a PR target gene we identified this gene in our signature the CTSD gene which is expressed in invasive breast cancer as a ER PR target gene you can see that PR comes here with estrogen and that we can also see the IGFR going away when we use the sumo mutant or phosphomimic PR and whoops I thought there was a circle there and we can see also that the IRS-1 can be found going up whereas the the receptor goes down the IRS-1 insulin receptor substrate one coming into the complex so we see the concomitant loss of the receptor at the gene whereas the the scaffolding molecule comes in there we can also see that so when we make these cells that are the sumo resistant or I'm sorry the the this the phosphomimic or sumo mutant cells become tamoxifen resistant so we can increase the concentration of tamoxifen and see that whereas cells that have the wild type receptor become sensitive are sensitive the cells with this phosphomimic or sumo mutant sort of an activated receptor you can think of it as they become TAM resistant in two different models and really interestingly and sort of the reason this paper probably got some attention was that these become exquisitely sensitive now to insulin so in two different models expressing these mutant receptors we can see that when we have the phosphomimic receptor the cells become very very sensitive to insulin if the receptor is the wild type or simulated receptor they're not very sensitive and here's another sort of TAM resistance in insulin and TAM so we think that this phosphorylation event is conferring this insulin sensitivity and TAM resistance in part by dragging in the IRS one we're not really sure why or what the IRS one is doing to the complex and we're still working on that but you can see also what happens in this situation as you see more stem cell markers aldeflor and CD 44 positive cells which are markers of stem cells in vitro and in vivo and those go up when we have the phosphomimic but are blocked when we mutate the serine and we can block their appearance and the appearance in tumor spheres by inhibiting the IRS one with a small molecule inhibitor or an inhibitor to the IR alpha receptor so we think what's happening is the IGF receptor is going away but the fetal insulin receptors is playing a role in breast cancer and this is really the work of Dr. Douglas Yee at our Cancer Center where IR alpha in the literature which is a fetal insulin receptor is getting overexpressed in breast cancer and we're losing the IGFR the IGFR this can also this biology also can be induced by growing cells in 3d and simply changing how the cells grow if you put them from 2d to 3d you see this phosphorylation event happens this is flow cytometry with our antibody in a regular cell in a TAM resistant model when they're put in 3d you see increased PR phosphorylation and you immediately see the expression of PR target genes that were in our signature stem cell mediators come up in the 3d and we so we think this is highly inducible upon PR phosphorylation and 3d conditions enable this signaling pathway we've also done chip assays that I'm not going to show you that can show the complexes present at all of these genes and these cells have stemless properties and that in that they grow in mammospheres even though you see very little PR here these are the TAM resistant models they grow ton of spheres they have a little bit of PR but the PR is inducible in this 3d situation we can also see PR target genes coming up in TAM resistance so these are IRS-1 and FOXO-1 which are fossil PR target genes that come up if we knock out the PR with shRNA we lose it and if we inhibit PR with antiprogestins and our IRS-1 inhibitor we can block tumor sphere formation and you can see that the T47D model is a little bit resistant these are your p53 mutant cells they behave a little differently and they have a really high AKT signaling so they're a little bit resistant but you can come in with ru46 and really blow that away here so combining an antiprogestin with an inhibitor of IRS-1 blocks the stem cell biology phenotype okay and so getting back to what could be happening and how this could be working we thought a little bit about what happens what do cells need for stemness and metastasis and they really need metabolic plasticity and if you look at really nice literature coming out from labs such as John Blenis's lab where he studies the mTOR signaling pathway and the idea that metabolic plasticity in cancer is really mediated by activation of mTOR 1 and 2 and AKT is needed and if you think about how often AKT is activated in breast cancer it makes a lot of sense and insulin is a really big input to this pathway and so we thought well let's just look at metabolism in our models and if you look at the downstream effects of this pathway they're all sort of altering metabolism and he has numerous review articles on this idea that cancer cells need metabolic plasticity in order to leave the primary tumor and and disseminate and so we took our went back to our PELP model where we know we have a ER PR PELP complex in in this this cytoplasmic complex I talked about and looked at those guys and they're highly metabolically active they have completely reprogrammed their metabolism and then using omics and different RPPA and different types of assays we identified this kinase in the complex this is a metabolic kinase known as phosphofructokinase and we found it in the complex it's also a PR target gene and if we knocked that out and did the MIND model this is where you put tumor cells right into the duct of a mouse and you can knock out the PFK you see very modest modest effects at the site of injection the primary tumors are trending to be less, but it's barely significant. However, if you look at the disseminated cells now and take the blood of the mice and look at the circulating tumor cell clusters, you can see the clusters are much smaller and there are many fewer clusters. So we have a really, really big effect on dissemination with a really modest effect on primary tumor growth, suggesting that we have different cell fates and different biologies that are happening. Okay, so kind of to summarize what I've told you, this biochemistry story that we tried to fit into this sort of insulin story is that you sort of have this big, ugly tumor, right? And as it grows bigger and bigger, it's gonna have a hypoxic center, high reactive oxygen species, neurotic core. It's gonna have nutrient deprivation. It's gonna be very cytokine rich. If you're talking about obesity and conditions such as this that are pro-inflammatory, that's gonna be even more rich in cytokines. And it's gonna drive this stemness program that I've defined. We typically target the ER in these primary tumors and that works very well. However, over time what happens is this stress-induced pathway can mediate EMT and raise the level of phospho-PR. And we probably need to think about targeting the progesterone receptor as well as metabolic mediators that we found in the complex. To eliminate those disseminated cells and reduce recurrence. Okay, and so this is my lab and all my collaborators who contributed to this work. A great deal of people added to this work. And primarily this was two post-docs in my lab, Amy Dwyer's first author paper and Thu-Truong, her collaborator in my lab. And with that, I will end. I'm quite early and I hope there's time for questions. Thanks. Thank you. Thank you, Carol. We have questions. Very elegant work, Marina Holes, New York Medical College. So as you know, IRS-1 at the membrane is very heavily phosphorylated by many growth factor signaling pathways. What does it look like when it's in your ER-PR complex? Yeah, we really wanna do that. So Amy did this beautiful work and then flew off to Australia to be in Wayne Tilley's lab and she's from Australia, so you can understand how she wanted to go back home. And we just haven't done it, but I'd love to do it. Because I think there must be something special about it and I think it's probably phosphorylated and maybe scaffolding more coactivators or different coactivators or maybe even remodeling complex members to get, it's going to different genes when it has IRS-1. So how is it doing that? So that's probably the key, right? That's a great question. Very nice work. I have two questions, actually. So I'm wondering with regards to your phospho-PR, I might be wrong, but it seems like tamoxifen is increasing the growth of the cells. Could this potentially be a mechanism of not only resistance but something we're not considering is that tamoxifen in this scenario might actually be increasing the growth of tumors? Yeah, I think it is. And I think we also see this, we see this dramatic increase in sensitivity of the cells to progesterone and anything driven by PR when they either are in tamoxifen or fulvestrant. This also showed up in Katherine Briskin's recent paper. This sensitivity is really high and I suspect what's happening is a stress pathway is coming on and it's causing these kinases to come up enough that it's getting these scaffolding molecules to form because of the phosphorylation events. And it's not just that. So that serine 294 is kind of a hotspot. It's a propolisomerase site. What we think is happening is getting phosphorylated and then a propolisomerase is recognizing it and twisting it. And that's a very large structural change. And we probably need Kendall to help us figure that out. So it's not just that site. It's numerous sites, but that site seems to be a really good marker for that. And so in tamoxifen, we definitely see that phosphorylation event, bam, come on. And then it's nucleating complexes. My second question is with regards to risk. So we've looked at it just very briefly, ERPR expression in the normal mammary gland. A number of people have shown mutually exclusive expression in some cases. In some cases you see both. I wonder how these signaling pathways are affected in the normal cells and whether this might be also an indication for risk. It might be. That's a great idea and a great question. I'll just say, I know that Christine Clark and Denny Graham in Sydney showed that when ER and PR are co-localized in the same cells, that it sort of tracks with a metastatic gene signature and an invasive gene signature with SART kinase, which makes a lot of sense. And that in normal tissues, they tend to be less co-localized. And then I will say that I worry about the use of the monoclonal in clinical samples because I think the heavily modified progesterone receptor is masking the epitope on the mono. And our poly shows, it's a really beautiful antibody. It's really clean. We've done all kinds of validation. It shows a lot of PR and we don't see any correlation between total and phospho, either in ovarian cancer, fallopian tubes, any kind of tissue. So we think that that monoclonal is not always accurate, right? And so that's scary. And I've heard that happens with the ER as well. Elaine Allered is doing some of that work too. Yeah, good to know. And maybe we can talk about. Thank you. So your differences between 2D and 3D might suggest there's mechanical transduction events that are potentially driving some of the effects. So have you looked at the role of FAC or whether the signaling events are different in the 3D environment? Yeah, we're doing that now. I suspect it's probably integrin type signaling or notch because that sort of requires the sort of the 3D structure where cells know which way is up and which way is down, which they don't know in 2D. So yeah, I'd love to know what that is. We don't know the answer yet. If it's a few signaling pathways or a particular one. I'm guessing it's integrin or notch. Just hit him. So could you, I mean, I feel like the, and I don't know if you've tried this yet. One of the money experiments would be like if you could take your phosphocyte mutants and see if that would impair like an obesity sort of hyperinsulinemia driven mouse model of breast cancer. And you know, actually the other thing is that you already have the antibody. You could probably interrogate somebody else that's already done this and if they have leftover tissues to see if the patterns are what you suspect. Yeah, that's a great idea. And in preparing for this, I mean, I was kind of surprised they placed me in this session to be honest. And I was like, oh my gosh, you know that. And then when I started reading about it, I thought, oh, we have to study this. You know, we have to study this. So I think you guys recruited me into the field to study breast cancer risk and in obesity. So that, yeah, I think that would be great to do. And the antibody works well and we have a lot of it. And so we're starting to look at clinical samples and also clinical samples from clinical trials of progesterone and antiprogestin treated women who have breast cancer, but we haven't gotten to the obesity group yet. I think that'd be great. And also I'd love to look at stress, life stress for not only PR, but GR. We have almost the same paradigm going with phospho-GR. They're very similar. So does GR have, is it basically an equivalent site? It has a P38 MAP kinase family member site in about the same place that is a complete input for TGF-beta. You do not need cortisol at all. You just put a drop of TGF-beta and you will get migration that's GR dependent and dependent on that site. And it brings in, same thing, scaffolds a bunch of molecules and goes to different genes. It's the same story. Just substitute GR and triple negative breast cancer wherever you saw PR in luminal. It's crazy. Great discussion. Thank you, Carol. Thanks, you guys. Thank you. So our next speaker is Dr. Christy Brown. She is at the Weill Cornell Medical College and the title of her talk is Obesity and Breast Cancer in BRCA1 and 2 Mutation Carriers. Thank you. And thanks to the organizers for this opportunity. It's always good to be in these sessions, but especially good this year as we can see everyone again. So we'll skip to the disclosure slides. I have nothing to disclose. My lab has been interested in trying to understand the molecular relationship between obesity and breast cancer for quite some time now. And this is because there's strong evidence linking obesity to the development of multiple types of cancer, at least 13 of them. And although the relationship is strongest with endometrial cancer, for example, hormone receptor positive breast cancer after menopause is still important given the number of breast cancer cases that we see every year. So it's no surprise that breast cancer could be affected by the fat and the adipose tissue and dysregulated metabolism, considering that the glandular tissue, which is the site of origin of these cancers is surrounded by adipose tissue. We also know that this adipose tissue changes substantially with obesity as it does in other depots, probably more similarly to the subcutaneous depots. So we and others have spent quite a bit of time trying to understand how the adipose microenvironment may affect the growth of cancers as well as the progression of disease. And it's been shown that with obesity, we see changes in the adipocytes that lead to an increase in secretion of leptin, for example, a decreased secretion of adiponectin. And when these adipocytes become dysfunctional and they die, they will lead, it will lead to the recruitment of immune cells, including macrophages that will infiltrate the tissue and surround these dead or dying adipocytes leading to these inflammatory foci. So obesity is really consequence or really leads to chronic low-grade inflammation, not only in visceral and other subcutaneous depots, but also in the breast depots. In addition to these cells, we also know that these various factors can act on the adipocyte precursors, the adipostromal cells, and they can stimulate aromatase expression within these cells leading to an increased production of estrogen and really creating a hormonal milieu that will support the growth of tumor cells. What is less well appreciated, I guess, is how this microenvironment may in fact also cause cancers to occur, so cause tumor initiation, because you'll understand that risk is really a combination of the detection of tumors that might be growing faster, but also causing genetic or genomic changes that lead to cancer formation. So we became interested in BRCA mutation carriers for a number of reasons. Individuals with BRCA1 or BRCA2 mutations have an increased lifetime risk of breast cancer relative to the general population. It's in the order of four to seven times what we see in women, and 50 to 85% of women who carry a BRCA mutation will in fact develop breast cancer. So this is a substantial number, but you can also appreciate that not all women are developing breast cancer with these mutations, so there must be modifiers that are involved in driving cancer formation in these women. Now these are DNA repair enzymes, so they're two very different enzymes, but they're involved in repairing double-strand breaks along the same pathway, and this pathway utilizes homologous recombination for DNA repair, which is quite an effective and reliable means of repairing DNA, and when these pathways are defective, cells will rely on alternate means of repair that will introduce potentially mutations or chromosomal defects that can lead to cancer. So we were interested in studying this population because, well, you know, this population has a defect, so we might be able to capture something that might not be so easy to see in the general population. So our initial idea was, okay, this is a nice model to study, but the flip side of that is really trying to understand whether these women who are at high risk with very few therapeutic or preventative options might be able to modify their risk if we're able to identify a modifiable risk factor as a driver of changes. So there have been a number of epidemiological studies suggesting a link between obesity and breast cancer risk in these women. The most important is probably the work from Mary Claire King and colleagues, who demonstrated that maintaining a healthy weight or being physically active earlier in life was associated with a decreased penetrance of both breast and ovarian cancer. There have been a number of other studies since then that have shown an impact of lifestyle as well as including diet and obesity on the development of cancer in these women. But equally, there have also been studies showing no effect. And I think it's important for us to start understanding what the differences are between these studies that will lead to a different interpretation of data. And perhaps going into a preclinical setting, we might be able to have a more controlled view of how this might be happening if it is. And if it is, the mechanisms whereby obesity is actually leading to an increase in cancer are still poorly understood. So in order to start asking this question, we accessed normal breast issue because we're interested in the initial stages of cancer formation from 72 BRCA mutation carriers in collaboration with colleagues at Memorial Sloan Kettering. We generated tissue microarrays, and this is just an example of one of them. So you see here, if you're not familiar, we were able to put a number of cores from tissue from different patients, and we had four of those. So we had 72 patients, multiple replicates, so representing various areas of the normal breast. We had women with BMIs ranging from 17 to nearly 45. The majority of the women, however, had a healthy BMI, what's considered healthy in this case, so 64% of them. We had 42 BRCA1s and 30 BRCA2s, and the majority of women were also premenopausal. And these women, we were able to obtain their tissue from either prophylactic or therapeutic mastectomies. So our question was, what might be happening in the normal epithelium in relation to obesity that could help us understand whether obesity has an impact on cancer development? And because these are DNA repair enzymes, we decided to look at the amount of DNA damage within this tissue. So Hiba Zahid, who was a PhD student with me when we were in Melbourne, was able to develop a quantitative assay to measure gamma H2AX foci, and if you know anything about this field, this is a very well-accepted marker of double strand breaks. So the way that this works is that when a cell detects a double strand break with ATM, which is a kinase, it will phosphorylate the histone H2AX, leading to a gamma H2AX signal that spans really about 100 kilobases on either side of this break. So with immunofluorescence, we're able to detect single breaks as these foci that appear. So this is an HNE of one of the cores, or part of one of the cores. You can clearly see the milk ducts, and this particular case is heavily surrounded by fibrous tissue and some adipose tissue. And when we convert this to an immunofluorescent image, we see the nuclei in blue, and it's always hard to show during these talks, but the foci of gamma H2AX come up as these very specific red foci. And we've developed techniques to be able to quantify this in an unbiased way using software analysis. So the work that I'll be presenting today is that of a grad student in my lab who's just finishing up, Priya Bhardwaj. So she spearheaded most of this project. What she found was that the number of these gamma H2AX foci were positively correlated with the body mass index of women from whom we had obtained the tissue. So this was suggesting that a higher BMI was causing more damage, or was associated with more damage, which may be a result of more damage or decreased repair, and that this may be accounting for a change in risk for these women. So to be able to start thinking about risk reduction and using this as a potential biomarker, we wanted to understand what the possible drivers of this DNA damage might be. So to do that, we looked at a number of circulating as well as locally derived factors. What we found was that a number of markers of insulin resistance and metabolic dysfunction were positively correlated with the amount of DNA damage, including insulin, glucose levels, and HOMA-IR score. What was, I guess, a bit surprising to us was when we looked at local inflammation via these crown-like structures, or we looked at C-reactive protein, or IL-6, we did not see that association. So it was suggesting to us that there might be differences between different patients as far as what might be driving this phenotype. Because my lab has been interested in steroid hormones, we also looked at SHBG. As you know, SHBG will bind bioactive steroid hormones, and what we were seeing here is that higher levels of SHBG were associated with lower levels of DNA damage within the breast glands. So we went on to do a couple of studies in MCF10A cells that have, sorry, heterozygous mutations in either BRCA1 or BRCA2, as well as primary breast epithelial cells from BRCA mutation carriers. And what we found was that insulin was able to stimulate the amount of damage, albeit modestly, within these samples, maybe a little bit more in the primary cells. So this was suggesting that hyperinsulinemia may in fact be one of the drivers that we were seeing here. But we were also curious to know whether the local breast microenvironment may be able to drive some of the effects that we were seeing. So to do this, we developed a couple of models. We developed a 3D co-culture model in which we cultured some primary breast epithelial cells, and you see them here as dots, but these are really cells that have grown over time in collagen and formed these acini with multiple cells. And then we're able to overlay these cells with adipose tissue or any other cell that we're interested in. And what we found was that in overlaying these cells with obese adipose tissue, we see a significantly higher number of DNA damage foci compared to when the cells are exposed to lean adipose tissue or collagen alone. So this was suggesting that the adipose tissue might be a driver of some of the effects as well. We also obtained conditioned media from reduction mammoplasties from women across a range of BMIs, and when we treated our BRCA heterozygous cells with conditioned media, we also saw a positive relationship between the amount of damage and the BMI, again supporting a potential role of locally derived factors. So to start teasing out which factors might be involved, we performed RNA-seq on breast tissue from BRCA mutation carriers. In this case, it was about 150 different women, and what you see here is the unsupervised clustering of gene expression with the lean cases in blue and the overweight and obese cases in red. So they did tend to, I guess, cluster together, but maybe not surprisingly, at least to us, we did see some outliers, if you will, so some obese cases clustering with the leans and some leans clustering with the obese, and this comes back to that idea that BMI might not necessarily be the best predictor of these groupings that we often talk about, and I think that it's really a reflection of metabolically obese lean individuals being clustered. So we're keeping this in mind as we're doing our analyses. We also looked at changes in gene expression within the various canonical pathways, and many of the expected ones came up, including immune-related and macrophage-related gene expression signatures, as well as estrogen biosynthesis, which we're particularly interested in, HIF-1 alpha signaling, et cetera. So this is not necessarily very different to what we see in women who do not have a BRCA mutation, so not necessarily surprising. But then when we started thinking about what are the differences in expression between the women who have a lot of damage versus not, irrespective of BMI, those same types of pathways obviously came up, but estrogen signaling end up having a stronger relationship than when we were looking at in relation to BMI only. We were also curious to know whether we could utilize information from breast epithelial cells to try to infer interactions between the breast glands and the adipose tissue. So we isolated the epithelial cells of the breast, actually based on a protocol that was published by Denny Graham and Christine Clark, whereby we can mechanically and enzymatically digest the tissue and isolate only the epithelium of the breast. And in that case, these organoids really maintain their structure. So we see here a basal cell marker that surrounds a luminal marker of the breast. So we're able to study these organoids outside of the microenvironment. And when we looked at the upstream regulators that may account for gene expression changes in lean versus obese organoids, many of the same factors came up, but estradiol was one of the top hits, as was insulin. So this was really reinforcing that there was a strong interaction potentially between the adipose tissue and these organoids, or dysregulated metabolism in the organoids. So the idea that estrogen exposure in BRCA mutation carriers is a risk factor is not novel. Most of the studies actually focus in this area, and there are a number of studies that suggest that this is important for both BRCA1 and BRCA2, even though BRCA1 mutation carriers tend to develop triple negative disease. One of those studies showed that risk reduction, salpingo-oophorectomy, so removal of the fallopian tubes and the ovaries, is associated with a decreased risk of cancer in both BRCA1 and BRCA2. Another study, or many other studies, have demonstrated that tamoxifen decreases the risk of contralateral breast cancer in both BRCA1 and 2. So we think that estrogen plays an important role in both mutation types. So to start asking this question, we first had a look at the tissue, and now I'm debating whether we should be looking at other antibodies as well, but at least we get an ER signal here. So we do see, this is normal human mammary gland, and we do see ER expression in both BRCA1 and BRCA2, and when we look at DNA damage here seen in green versus ER alpha in red, we do see some colocalization of these signals in the same cells, or at least, you know, the colocalization at a cellular level. We're still not sure whether there's an actual interaction between the two, and we're interested in this as well. So on the back of that, we went back to our organoid model, and we decide to treat these with estradiol, and we do see an increase in DNA damage in these, and that's reversible with fulvestrin, suggesting that the effect is mediated via the estrogen receptor. Because my lab, you know, has a longstanding, I guess, interest in aromatase regulation in the breast and the role of aromatase in driving breast cancer risk, here's an example of aromatase in the breast of a post-menopausal woman with breast cancer. You see in brown that, you know, the expression is very high adjacent to a tumor, and aromatase, as a consequence, is able to support the growth of these tumor cells. But we've also demonstrated that aromatase levels are positively correlated with BMI within the breast tissue. So with higher BMI, we see more aromatase on a per-cell basis, and this may also be driving risk in many women. So we looked at the relationship between DNA damage and aromatase, and again, we do see a positive correlation. We think that this is probably one of our strongest associations out of the markers that we've seen so far. And to start asking whether, you know, the microenvironment is sufficient to cause this, we also treated breast tissue explants. So using, actually, Wayne Tilley's model that he presented yesterday, where we culture tissue on top of surgical sponges, we treated these tissues with fulvestrant. So in the absence of exogenous estrogen, and we're able to suppress the amount of damage in these tissues, suggesting that there's an active source of estrogen within the tissue. My lab, a while ago, had also shown that metformin was able to suppress aromatase expression in the breast adipose tissue. So we were curious to see whether we might be able to start thinking about interventions that are feasible in a patient population. We wouldn't want to be giving fulvestrant for decades, you know, to reduce risk, but metformin is something that's relatively well-tolerated. So we looked at the effects of metformin on DNA damage, and interestingly enough, we saw a really nice dose-dependent effect of metformin on the amount of damage in the tissue explants, but not so much in the isolated breast organoids. So this was suggesting that the effect was dependent on the microenvironment, and potentially effects on aromatase, although we know that metformin will act on other systems as well. So to test that question of the effect on aromatase, we collaborated with Man Ho Choi, who's actually here today, and we were able to measure steroid levels within the tissue. And what was nice was that, and I think this is the first time that we actually look at steroid levels, so it was a nice proof of concept, was that metformin caused a dose-dependent decrease in estradiol levels in the tissue, but a concomitant increase in testosterone. So this was suggesting that we were hitting the conversion, potentially, of testosterone to estradiol. So we were mindful that we didn't want to give all of the credit to estradiol, so we also looked at effects of other adipose-related factors. This is the effect of leptin. Again, we're seeing leptin stimulating the amount of damage in both MCF10A cells, which in fact don't express the estrogen receptor, as well as in primary BRCA mutant epithelial cells. And interestingly, if we use a leptin-neutralizing antibody, we're able to reverse some of the effects of the conditioned media, suggesting that we might also be able to intervene at this stage. So to start being able to ask questions of interventions without going into a patient population just yet, we developed a mouse model of diet-induced obesity on a BRCA1 heterozygous background. So these mice hadn't been studied yet because most of the BRCA mice are not on this background. So we were able to back-cross existing mice, and when we fed them either a low-fat or a high-fat diet, we saw what was expected, an increase in weight. So mind you, these are ovary-intact mice. We're still debating whether local production of estrogen in this model is relevant, considering the differences in aromatase expression. So we wanted to maintain gonadal steroid production. We also wanted to mimic the patient population as much as possible. So these mice do gain weight, and they do develop some glucose intolerance. And as expected, they also have an increase in mammary fat pad weight. And if we compare gene expression, so here it's a little bit obviously hard to see, but we have our high-fat mice all the way on the right and our low-fat mice in between. And the first column there is the changes in gene expression in the human. So we were trying to compare changes in human gene expression with what we were seeing with high-fat feeding, and there's a really nice relationship between these. And this suggests to us that this model may in fact be useful to predict some of the effects. So when we looked at DNA damage in these mice, we did see a significant increase in the amount of damage in the mammary gland that tended to correlate better with the mammary fat pad weight relative to the body weight. Again, emphasizing that body weight is not necessarily everything, and BMI might not necessarily be the best measure of effects that we're seeing. So to start asking the question with regards to tumor formation, it's hard with these models because we're expecting that diet and obesity and metabolic dysfunction are predisposing to cancer formation via effects on DNA damage. So we modified the MPAD-MBA model really to reduce and minimize the amount of carcinogen we were using so that we could actually use it as a modifier as opposed to a regimen that would induce tumor formation in all of the mice. And what we found was that 86% of the mice on high-fat diet developed tumors compared to 69% on low-fat diet. And these mice were also developing tumors earlier than the mice on low-fat diet. So all of these findings were really suggesting that obesity can modify DNA damage in the human and the mouse mammary gland as well as tumor formation. So there might be an opportunity to intervene and reduce risk in these women. I'd like to show this just to emphasize that obesity and adipose tissue is no longer just a driver of cancer growth and metastasis, but it may also be really important as far as tumor development. And I mentioned Priya. She's been an exceptional scientist and will do great things, I'm sure, in the future. Other lab members as well who were involved. We worked with Lou Cantley to obtain the mouse model. Our bioinformatics colleagues, Ashley and Olivier, our clinical colleagues, both at Weill Cornell and Memorial Sloan-Kettering, as well as Michael Polak, who did the blood biomarker analyses, and Man Ho, who I mentioned as well. Our funding and, of course, the women who donate their tissue to us multiple times a week without whom we wouldn't be able to do these studies. Thank you. All right, we'll start questions. It looks like there's none online yet. So, Katya, you want to start? Katya Kisek, Vassiliales University of Colorado. Great talk. So, this BMI story is a little bit all over the place. So, I was wondering whether people have looked at better metabolic risk factors, diabetes, metabolic syndrome, or actually PCOS. Is there any data in PCOS women? And we know that there's a lot of data in PCOS women, but is there any data in PCOS men? PCOS women, and we know that we use them in the form of PCOS women, so some of the women with the scarier gene. The PCOS question has always fascinated me as well. And I don't know that there's strong evidence one way or another, because I think PCOS is so different, right, across all of the patients. And I'm not sure, actually, many of you would probably know better than I do with regards to whether it's been looked at to segregate, you know, the women who have high aromatase versus the ones who don't. Or, you know, those differences may actually have a huge impact on risk. With regards to BMI, I think we're still trying to figure out which risk factors that are associated with higher BMI are the modifiable, are the ones that we can start tackling from a risk reduction point of view. Because we know that with obesity, you know, you get insulin resistance. That's associated with an increased risk of cancer on its own, so independent of BMI. So it might be one of the major drivers. But high, you know, steroid hormone levels as well as associated with risk, independent of BMI. And when you're thinking about interventions in an obese population, it's still not clear which one of those, if not all, need to be reduced in order to reduce risk. All right. Matt Sikora, Colorado. Beautiful stuff, Christy. So do you only see the obesity phenotype and the signaling driving increased damage in the hemizygous context? I ask because I'm wondering whether those cells are actually haploinsufficient to deal with stress or whether you're hastening progression to LOH. That's a great question. And, you know, we've had the question of, does this happen in wild-type cells? And does this happen, so part of a funding application that's been submitted and is hopefully under review now is to look at what's happening in the tumor as well. Because that will change the course of treatment as well, right, if you can start, you know, factoring that in. Without going into a lot of detail, we do also see effects on DNA repair enzyme expression in relation to obesity. So part of us is thinking that obesity is maybe driving that second hit that you would expect to need to have things progress. But it's still early days with regards to that. But that's a great question. So in your IF data, maybe, are the cells that are H2AX positive, are they BRCA1 or BRCA2 null by protein? It's just been tough for us to measure that. If anyone has any good antibodies for that, let me know. But it's just been tough. We've done it, we've tried to do it by Western as well. That's been particularly hard. Thanks. So with your readout of gamma H2AX positivity, does that reflect increased genesis of DNA damage or does that reflect reduced repair of the DNA damage because you're catching them at one point in time? And can you take advantage of your cell line and organoid models to do time courses and also measure how long it takes for those marks to resolve to get at the mechanism by which insulin, estrogen, leptin can cause that increased signal? Great question. That's the focus of our R01 renewal and also the focus of one of the grad students' work. So what I can say is we did do some, we looked at resolution of foci after radiation, but in the context of conditioned media treatment. There we didn't see a huge effect. I think we did it with leptin as well. But I'm still not convinced that it's not happening given the effects on gene expression that we were seeing within these pathways. So we just want to go back to our, it's tough working with the primary cells, as you can imagine, right? So we're trying to optimize those models to be able to answer those questions in a better scenario. We've also done it with X-plants. Actually, that might be where we need to go. Yeah. Really, really lovely and also really hard to do. You probably know this, but so BRCA carriers have elevated estrogen because their ovaries kind of overfunction, but they have extremely high progesterone. And that's also been shown in mice as a defect in the, when you make a BRCA mutant model, that's all body. Their ovaries way overproduce estrogen and particularly progesterone. And so I was wondering if your BRCA models were mammary gland conditional or all body? That's a good point. So for us, they're all heterozygous mutants. So many in the field will actually associate that with no effect, right? So loss of, I'm sorry, haploinsufficiency has been suggested at a cellular level, but not necessarily in patients. So as far as, you know, showing an effect. But I did not know about the progesterone side of things. So it's a global heterozygous knockout. The interesting thing that probably plays to what you were saying as well is we do head-to-head breeding and we never see knockout mice. So there are obviously big issues when you have a global knockout. I bet you have the ERPR complex happening in your model. I'd be happy to work together. We have time for one more quick question. So is the DNA damage you're seeing replicative stress? Is there correlations with damage and proliferation? Yeah, that's part of what we're exploring at the moment. It's not 100% clear. So we looked at various markers of proliferation, Ki67. You know, but it's part of Katie's ongoing work at the moment to determine that. That was her underlying hypothesis. We have also seen effects on ROS production. So it may also be coming from somewhere else. Thank you, Chrissy. Thank you. Thanks. Our final speaker is Dr. Elizabeth Wahlberg from University of Oklahoma. And she's going to tell us about the impact of weight gain and FGF1 on estrogen receptor signaling in breast cancer. Hi, everyone. Thank you so much for staying till the end. My talk is actually a little bit shorter than I thought it was going to be. So hopefully, we'll get out of here and make it to the airport in just a few minutes. Thank you guys so much for inviting me to give this presentation. I'm really, really excited to be here in person. So one of the things that I study, like the other speakers, is ER-positive breast cancer and obesity. And I do a lot of preclinical work. So I have no disclosures. And I was told to put that slide in there. Whoop. This is mine. Whoop. This is mine. Yep. Nope, we're good. So we all know obesity is prevalent. These data are relatively recent data from just a few years ago from women showing the global obesity prevalence. And you can just tell by looking at this that many countries have a pretty high prevalence of obesity. And it's projected that obesity will continue to increase in prevalence. And so when we think about studying cancer, I think it's important to incorporate aspects of obesity into our model systems. So obesity is, as we heard earlier from Christy, a risk factor for 13 different cancer types. This is an infographic that was put out in 2020 from NCI. And what matters the most to me right now is postmenopausal breast cancer. And I included this little handy pie chart, but I know this audience doesn't need this information. Most breast cancers are ER positive, and this is the subtype in a lot of the clinical literature that's been associated with obesity. So this is what I study in my lab. As we've also heard from Christy and from Carol, BMI isn't maybe the best way to evaluate cancer risk in people. So there are other things that we can use, such as metabolic health, which is a bit of a vague term, but it basically refers to insulin resistance, hyperinsulinemia, dyslipidemia, and that kind of thing. Weight gain is actually an independent predictor of breast cancer risk separate from obesity, and this is what I focus on. Adipose tissue expansion is a growth process, so there's a lot of growth factors that are produced. And adipose tissue distribution and health is another nice predictor for breast cancer risk that might be separate from BMI. So people with what's called central obesity have a higher breast cancer risk in some cases. So the way that I study this is in mice. Because mice don't typically produce ER-positive tumors, we use PDXs and human cell lines. And so we have to use immune-compromised mice. We use RAG1 null females on the C57 Black 6 background, and a high-fat or low-fat diet to separate the mature, obese, and lean phenotypes. The high-fat diet's about 40%, so it's not really high, but it's enough. But what really helps us achieve obesity in this model is warming the mice. We use thermoneutrality, where we basically house the cages partway on warming blankets, and the little higher temperature in the cages is sufficient to really help these mice gain weight. Once they're mature, we OVEX them and immediately supplement them with estrogen and implant tumors. And we do this this way because, first of all, estrogen is required in most cases for these human ER-positive tumors to grow. But we want to really study this estrogen withdrawal period, which is very metabolically disrupted. So once the tumor's established on estrogen, we can just, and we use drinking water estrogen, we can just change their water, and then you get a really nice experimental period of rapid weight gain that is something we're interested in. So glucose increases, insulin increases, adipose tissue changes dramatically. And so this is the model that we use for most of our studies. So just to give you sort of an idea of what happens, we've done lots of different studies now, but, and this is some work we published a few years ago, if we look at the change in tumor volume from before to three weeks after this estrogen withdrawal period, we see that some tumors do progress in the obese without estrogen, but not all of them do. For example, MCF7 cells, when they're injected into the fat pad and you take estrogen away, in this model, they don't continue to grow. So they're very estrogen-dependent. And just to show you what happens during this three-week period, these are pre and post-body mass measurements in lean and obese mice. So the tumor progression in some cases is associated with weight gain in the context of obesity. Of course, the first thing we were interested in is this estrogen, and these are mice, and I'm probably gonna catch some trouble from some people, but we are unable to show that mice produce estrogen in their adipose tissue after OVEX the way that humans do. So we've tried mass spec of the tissue, we've looked by PCR, and then we all use serum ELISAs, and we're just not able to convince ourselves that the adipose-derived estrogens are responsible for our tumor progression phenotype in mice. In humans, that's absolutely the case, but not in mice. So what does, what is driving the tumor progression? So as I mentioned, adipose tissue expansion is actually a growth process, and so there are a lot of growth factors in cytokines that are produced that are a little bit more higher risk than just stable obesity. So one of these proteins is FGF1, and it's actually very important for what's called healthy adipose tissue expansion during diet-induced obesity. So when you have a positive energy balance, your adipocytes get larger, and then the stromal fraction receives signals to proliferate so that those cells can differentiate into new adipocytes. And FGF1 is produced by really hypertrophic adipocytes through a really cool mechanism that involves physical tension from those cells, and then it's like this little channel opens and spits out FGF1. And then it signals to the stromal cells to grow, fibroblast growth factor. And so when we were starting this study, we looked at publicly available gene expression data of breast tumors from women who are classified as non-responders or responders to aromatase inhibitors, short-term aromatase inhibitors, and cross-referenced that with tumors from people with known BMI, women with known BMI. And then just looked at the inferred upstream regulators of those gene expression programs, and FGFR was one of the putatively activated signaling pathways. So in our mice, we find that FGF ligand increases in the adipose tissue. This is mammary adipose tissue, and increases during estrogen withdrawal in the high-fat-fed females. Again, that associates with the weight gain. The adipocyte size distribution shifts to the right, so these cells are getting bigger and larger diameter. And then on a individual level, the expression of FGF1 in the adipose tissue correlates with the rate of weight gain in grams per day during that three-week period, and with the average adipocyte diameter. And we've shown that FGF1 in the JCI Insight paper, we showed that FGF1, the same sort of pattern, takes place in human tissues as well. So in the tumor side, in human tumors, this is through a collaboration with Dr. Ann Thor, my former chairwoman in Colorado, we found that high levels of phospho-FGFR1 are associated with a poor prognosis for long-term follow-up for patients. And then in a different cohort, the same thing, we see high phospho-FGFR1 associates with an elevated BMI, but total levels don't necessarily associate with BMI. So in our animal PDXs, we saw that FGFR1 phosphorylation was elevated in the context of obesity, and if we treat the obese females with an FGFR inhibitor, we're able to restore sensitivity of those tumors to estrogen deprivation. So in a separate model, this is actually a paper that was just accepted like a week and a half ago, this is a rat model, we've done some of the same sort of things, so we're focused on weight gain, and so we use another MNU-induced mammary tumor model in rats, they do produce ER-positive tumors, and this is in collaboration with Paul McLean at the University of Colorado, who was one of my mentors during my postdoc. And we asked the question, so when we put these rats on a high-fat diet, all of them are on a high-fat diet, and because they're outbred, they separate into obesity-resistant and obesity-prone phenotypes, so that's where we get the lean and obese, but they're all on the same diet. And when you put them through overectomy, everybody gains weight, lean and obese, which is very common with estrogen withdrawal. So we just asked the question, if we prevent that menopausal or OVX weight gain, so they stayed on the same diet, they maintained their OVX BMI, or whatever you wanna call it in rats, their body mass, and we just gave them enough food so that they just didn't gain weight. And that was sufficient to delay the time of progression of the existing tumors, so these rats go into OVX with existing tumors, but they also form new tumors after OVX because the carcinogen has done its damage. So in both lean and obese animals, preventing weight gain delayed existing tumor progression and also prevented existing tumors regressed and fewer new tumors formed. And FGF expression was reduced during weight maintenance, is what we call this, in both lean and obese, and in the tumors, FGFR phosphorylation was reduced in the obese. So there's a lot of other stuff going on in this paper. Erin Giles is on it as well, and so hopefully that'll be out pretty soon and you all can read all about it. But so how then does FGF signaling impact breast cancer? And this is, my work is very much inspired by the women that we've heard from earlier, especially Carol Lang. When I was a postdoc, I loved her program on crosstalk with growth factor receptors and steroid hormones, and I think it's actually a very important paradigm and I think it explains a lot of obesity-associated cancer. So the simple hypothesis is that, of course, we know estrogen activates its receptor and causes tumor growth. But when you have estrogen deprivation, you get adipose tissue expansion. We have some other work focused on adipose tissue growth as a sort of side effect of cancer therapy and diabetes risk as a later consequence. So we hypothesize that FGF-1 just simply takes over to maintain the tumor dependence upon estrogen receptor, but not on estrogen itself. And that's a problem because the treatment for these women is typically to deprive them of estrogen. So if there's something else that's taking over, you're not gonna be very successful. This is not a new idea. As I said, there are a lot of people that study this and so I'm still learning about ER phosphorylation and all the different ways that that impacts its activity. And so for these studies, the rest of the data I'm gonna present really quickly is just focused on serine 118. I know it's not the only way that ER is activated, but it is the best we could do with the existing antibodies. So we're still working on some proteomics to try to get a better idea of what's going on. But basically, oh, so if you look in human studies, the high levels of phosphoserine 118 ER are, this is all in ER positive tumors, is a poor prognosis, but total levels are not associated with outcome in humans. And in our PDX tumors, we see elevated ER phosphorylation in obese, but no difference in total. And also the increase in ER target gene expression suggesting that it is in fact an activated receptor. So what we've done is experimentally go back into cell lines. We have the PDX cell lines from the University of Colorado from Carol Sartorius's lab. And we've also used standard MCF7 cells. So MCF7 cells, if you remember, don't grow in the obese. Estrogen increases the activation or the phosphorylation of its receptor, but FGF1 really doesn't do anything. But the UCD12, the cell line derived from the PDX that did grow in the obese, both estrogen and FGF1 can increase the phosphorylation of ER. And so because this is a really relatively new cell line compared to MCF7s, we wondered, we've had a lot of questions about why this happens and tested several things. But one of the things that we wondered about is just what if we look at a more similar cell line to the MCF7s? So we got the tamoxifen-resistant MCF7s. And in that case, under the same circumstances, estrogen doesn't really do what it's supposed to do, but FGF1 does increase ER phosphorylation. And when we put these TAM-resistant cells back into lean and obese mice, and then we take away estrogen for three weeks, the obese mice continue to support tumor growth of the tamoxifen-resistant cells. So we are now asking, what does FGF1 do through ER? That's something that I'm really interested in is the effect of, or is the role of ER in a tumor different depending on what activates it? And so we've done some RNA sequencing, and this is a lot to look at, but we were looking for a pretty specific pattern. What I wanted to see was the gene profiles, the genes that are associated with estrogen-mediated ER treatment, and blocked by ICI, which is fulvestrin, or had the same pattern with FGF1, and then blocked by ICI, suggesting that they might be estrogen-receptor-dependent FGF1 target genes. And so when we pull out those genes, there's a lot of other things that FGF1 does that doesn't depend on ER. But when we pull out the ER-dependent genes, oh, this is just another way of looking at it. So there actually are quite a few genes that are reversed with fulvestrin in the context of only FGF1 in the TAM-resistant, but if you look at the parental cells, that's not really the case. There doesn't seem to be a lot going on through ER in just normal MCF7 cells. And so if we look at the genes that are changed, that are going through ER, the top pathway is glycolysis and gluconeogenesis. And so this is kind of where we are now with the project, is moving forward and looking at how does estrogen receptor become a regulator of glycolytic metabolism, and what is the consequence to the cancer cells in the context of obesity. So these are just some of the genes that we pull out, and if you look at a heat map and compare the normal MCF7s to the TAM-resistant ones, you see that in the TAM-resistant ones, you begin to get this pattern of estrogen-dependent regulation of these genes. And I think that's really cool, and I'm really interested to learn more about how ER behaves at the chromatin level. So hopefully we can figure some of this out. And so, in summary, adipose tissue in lean and obese situations is pretty different, and it responds differently to stress. One of the stresses is a positive energy balance. The lean females, the cells get bigger, the stroma proliferates and maybe differentiates a little bit, but it's generally not a big problem. But in the context of obesity and a positive energy balance, you get elevated production of growth factors from these giant cells to just simply try to maintain the tissue function. And as we heard from Christy, the breast adipose is where tumors grow. And so if there's a tumor in there, it's just gonna benefit from the growth factors the same way that the stromal fraction would. So one of the ways that we think that this happens, this does its job, is through estrogen-independent ER activation in the context of obesity, and potentially by promoting an aggressive metabolic phenotype. And with that, I would like to thank my brand new lab. I just moved, it's not even brand new anymore. I've been there two years, but two years don't count. The last two don't count. But I have a really great lab, and I have really great former help, like students and technicians from Colorado, great collaborators, and still wonderful people in Colorado, and lots of good support. And thank you all again for sticking around till the end. All right, while people are coming up for questions, I'm gonna ask you one. In your different models, what do you see if you put in a triple-negative PDX? That's a good question. Since you're talking about ER. Yeah, well, and so triple-negative breast cancer in premenopausal women is associated with obesity. And so we've done, we actually have a study where we've done three diets. We do the low-fat and the high-fat, but then we have this low-fat, high-sugar, and I'm trying to figure out this insulin thing, insulin and glucose, like is that sufficient to drive these tumors? The triple-negatives don't care about any of it, but the ER positives appear to really, the triple-negatives grow like crazy regardless, but the ER positives, you know, just they like the sugar and they like the high-fat better. So yes, lots to explore. Super cool, so did you ever look at other FGFs, FGF2? Does it do? FGF2 didn't change in this model system, but it actually did pop up in the rat study, it's in circulation. So FGF1, we've not been able to measure it in circulation, but two does show up in circulation, but doesn't change in the adipose tissue. So beyond that, we didn't really go through all the endocrine FGFs, I mean, there's a lot of those that change with obesity as well, but no, we sort of just zeroed in on FGF1. Yeah, the reason I ask is that I think Claudio Nari has shown that FGF2 induces PR expression and activates PR. So it'd be like, oh. Oh yeah, yeah, yeah, that's right. Yeah, so I'm just wondering like why FGF1, but that makes sense. Well, because that's the one that was different, honestly, so we chased it down. And, you know, it is really, it seems to be really uniquely produced during the adipocyte hypertrophy. And I'm not sure FGF2 is produced that way, so yeah. But I absolutely, that's not the only thing, there's tons of things I'm sure that crosstalk, with as you, yes, you know. So what can you tell us about the signaling states of your cells? I'm trying to, whether I need to be more specific. So, you know, what do we know about when you add FGF, what happens to the signaling states in these cells? Yeah, they respond, MAP kinase pathway gets activated, AKT gets activated. We've done some proteomics on the parental and the TAM resistant MCF7s. And it seems that the, let's see if I can remember this, MAP kinase pathway is more basal, it's just on kind of more in those TAM resistant cells. And the AKT signaling is a little bit less stimulated. So one thing to keep in mind is, particularly for MAP kinase, is measuring phospho-MAP kinase doesn't give you flux through the pathways. Okay. So there's, you know, we can maybe talk about that if you want, but you may not, just by measuring the phospho states, you don't often get flux through the pathways. That's what's different between your MCF7 parentals. Yeah, and this is just one time point too. You know, I don't know what happens like over, I don't know what happens at, you know, one minute or even longer than what we did here, which was like a 15 minute time point. So yeah, I'm interested to learn more about how these cells behave. What was the genetics of your transcriptomics that you did? That was 24 hours. Yeah, so that was, we tried to catch a little bit of stable effects. Yeah, thank you. I have a question actually. So these cells sitting in a very fat rich environment, do they just simply up glycolysis and down regulate fat metabolism or? I don't know that they do that, that clearly. I think that, so in the rat we've used tracers and they seem to do everything better in the high fat. They, this like concept of metabolic flexibility that we heard about earlier. They can use fat, they can use sugar. And so one of the things I'm interested in is this concept that maybe the metabolites themselves may be changing the cell fate. And so we're looking to do some metabolomics in the future, near future, hopefully. Sounds great. We don't have any other question. Okay. Well, let's give another round of applause. Thank you.

obesity

BRCA mutations

breast cancer risk

DNA damage

insulin

glucose levels

insulin resistance markers

local inflammation

SHBG

hormones

cell models

co-culture experiments