Skin Stem Cells: Their Biology and Promise for Medicine
Transcript of Part 3: Cancer: Hijacking the Wound Repair Mechanisms Used by Stem Cells
00:00:14.02 My name is Elaine Fuchs. 00:00:15.24 I'm a professor at Rockefeller University in New York City, and I'm also an investigator 00:00:21.04 of the Howard Hughes Medical Institute. 00:00:23.23 All of the tissues of our body must be able to replenish dying cells and repair wounds. 00:00:30.25 We cannot get rid of those fundamental mechanisms; they're essential to life. 00:00:36.19 And yet it's those mechanisms that cancers hijack in order to take advantage of... 00:00:44.01 of, effectively, the ability of our stem cells to be mobilized and repair wounds, 00:00:49.22 this time doing it uncontrollably. 00:00:54.23 So, we study specifically squamous cell carcinomas. 00:00:59.08 These are the second most common cancer worldwide. 00:01:05.09 When we consider that squamous cell carcinomas are not only found in the skin, 00:01:10.04 but also head and neck, cervix, esophagus, cancers of the anal-genital tract, and some types of lung, 00:01:18.19 breast... and breast cancers, as well as skin cancers, now we're talking about 00:01:22.26 the sixth most deadly worldwide cancer. 00:01:27.09 And yet there's very few effective therapies for squamous cell carcinoma treatment. 00:01:34.12 So, what we've learned in the course of the field's studies on skin stem cells is that 00:01:43.18 squamous cell carcinomas of the skin can originate from skin stem cells. 00:01:49.22 They're there for the long haul, so they can acquire the mutations that ultimately 00:01:55.09 will lead to cancers and tumors. 00:02:00.06 So, some years ago, my laboratory isolated and characterized the tumor initiating cells, 00:02:08.19 the so-called cancer stem cells, if you will, that are giving rise to squamous cell carcinomas, 00:02:15.00 in this case using our favorite model system, the laboratory mouse. 00:02:20.23 The way in which we examined this was to generate a mouse that we could effectively 00:02:28.24 create by chemical carcinogenesis or by genetics skin cancers or squamous cell carcinomas. 00:02:36.24 And we isolated those squamous cell carcinomas, fractionated their cells using 00:02:42.18 fluorescence activated cell sorting, and then performed serial transplantation of each of the pools 00:02:49.02 that we isolated from the FACS machine, tested them one by one, individually, 00:02:55.02 to find out which one or ones had activity that would lead to the generation of 00:03:02.00 a new squamous cell carcinoma in a host recipient mouse. 00:03:06.26 We got this down to almost the near single-cell level, where we could take a single cell isolated 00:03:13.12 from a squamous cell carcinoma and introduce it into a host recipient mouse, 00:03:19.01 and get a squamous cell carcinoma. 00:03:21.19 That told us that we had a so-called cancer stem cell. 00:03:25.08 What does it look like? 00:03:26.20 So, we learned that the cancer stem cells produce a new set of transcription factors 00:03:33.05 that are different from the normal stem cells. 00:03:36.05 So, the cancer stem cells produce ETS2, ELK3, AP1, KLF5, and SOX2 and 9, 00:03:44.00 whereas the normal hair follicle stem cells produce Sox9, Tcf4, Tcf3, Nfatc1, Nfib, and Lhx2. 00:03:55.08 There's very little overlap in the transcriptional regulators between the hair follicle stem cells 00:03:59.21 and the cancer stem cells. 00:04:02.28 One of the interesting new sets of genes that are important in the squamous cell carcinoma 00:04:09.25 cancer stem cells are ETS and the ELK binding sites. 00:04:14.27 These are present in nearly all squamous cell carcinomas -- super-enhancers, those are the 00:04:19.21 regulatory elements that are controlling the activity of the stem cells -- and those factors 00:04:26.26 turn out to be phosphorylated and superactivated by a particular kinase, the MAP kinase 00:04:33.10 that is downstream from, in this case, oncogenic RAS or hyperactivated RAS, 00:04:39.08 which is frequent in squamous cell carcinomas. 00:04:42.25 So, the Ras/MAP kinase pathway, then, phosphorylates and activates ELK3 and ETS2. 00:04:50.14 And we don't yet exactly know how it works, whether it recruits histone acetylases, 00:04:56.19 or whether it increases the DNA binding affinity or the nucleosome binding affinity for the transcription factors, 00:05:03.27 but what we do know is that the phosphorylation and superactivation 00:05:09.13 of these transcription factors is critical with regards to altered, 00:05:14.27 malignant program of gene expression produced by these stem cells. 00:05:20.08 And we can also take advantage of cloning one of these regulatory elements that is 00:05:26.00 turned on in the squamous cell carcinoma stem cell. 00:05:29.15 And we can use that to express a GFP -- fluorescent green protein -- in mice. 00:05:35.28 And we just use a fluorescent red protein as a control. 00:05:41.10 But what we find is that the regulatory element, now, is specifically expressed in 00:05:50.22 the squamous cell carcinoma cells. 00:05:54.01 So, we know that the skin stem cells from squamous cell carcinoma originate from 00:06:00.05 the wild type skin stem cells. 00:06:02.07 But they don't look anything like the wild type skin stem cells. 00:06:07.25 They have a completely different program of gene expression. 00:06:11.09 They're expressing elevated levels of cyclins, which force these cells into a superproliferative state. 00:06:17.21 They express transforming growth factor alpha, a growth stimulator 00:06:22.22 for the squamous cell carcinoma cells. 00:06:25.22 They express certain cell survival genes that are long-term survival genes not normally 00:06:31.13 expressed by the hair follicle stem cells or by the epidermal stem cells. 00:06:35.22 They express genes which give... which are important for epithelial-to-mesenchymal transitions, 00:06:43.14 for invasion. 00:06:45.16 They express VEGF-A that recruits blood vessels to these stem cells, and so on and so forth. 00:06:54.25 These are, effectively, a list of genes that are implicated in many types of cancers. 00:07:01.26 And yet they're not properties of normal stem cells. 00:07:05.25 So, the cancer stem cells, as we're learning, reside at the tumor-stroma interface. 00:07:11.25 And that's very similar to what I described to you for the normal stem cells. 00:07:17.03 Epidermal stem cells and hair follicle stem cells reside at the epidermal-dermal interface. 00:07:23.15 And so too, now we're finding that squamous cell carcinoma stem cells reside at the interface 00:07:29.20 between the epithelium and the stroma. 00:07:31.24 But, now, the stroma is entirely different. 00:07:35.06 There are immune cells. 00:07:36.25 There are altered fibroblasts. 00:07:39.01 There are blood vessels. 00:07:41.10 There are a whole variety of different changes in the tumor microenvironment that 00:07:47.21 distinguish the stem cell microenvironment from its normal counterparts. 00:07:53.07 So, there's heterogeneity, also, that arises in the tumor microenvironment, and that turns out 00:08:00.03 to influence their behavior. 00:08:02.20 Wherever there is a blood vessel that comes up next to the tumor-stroma interface, 00:08:09.02 those stem cells, now, respond very differently, because the perivasculature contains a growth factor 00:08:15.22 called transforming growth factor beta, which is a member of the BMP superfamily 00:08:21.14 of signaling factors. 00:08:23.14 And now that signaling crosstalk between the perivasculature and the cancer stem cell 00:08:30.05 changes the properties of the cancer stem cells. 00:08:33.04 And it does so in a very important way. 00:08:35.26 First, the stem cells become slower cycling as a consequence of receiving this growth factor. 00:08:43.05 But more importantly, these stem cells become invasive. 00:08:48.26 And that is a very nasty thing. 00:08:51.02 So, in order to be able to study this, we engineered a mouse to mark the TGF-beta-sensing 00:08:57.12 stem cells of the tumor in red. 00:09:00.20 And we also engineered the mouse so that we could track the behavior of these TGF-beta-sensing stem cells 00:09:09.24 by activating them and their progeny -- following their progeny -- in green. 00:09:15.18 And so as you can see from the tumor, here, the green cells, the progeny, of the TGF-beta... 00:09:22.12 of the TGF-beta-sensing cells became invasive, and broke down the basement membrane of the tumor 00:09:30.26 and started to invade the stroma. 00:09:34.19 What's interesting is that this stroma is where the blood vessels are that 00:09:40.04 basically prompted this initiative effect. 00:09:42.13 And so we think that it's these cells that are then the ones that get into the bloodstream 00:09:48.07 and could be the roots of metastasis. 00:09:53.08 Another interesting and important aspect from these studies turned out that the TGF-beta-responding 00:09:59.16 stem cells have an increased resistance to chemotherapy. 00:10:03.13 So, the drug of choice for treating squamous cell carcinomas of the head neck is cisplatin. 00:10:10.17 And here we treated the tumor with cisplatin, and by day 3 you can see the blue cells, 00:10:16.27 which are the dead cells, are the majority of the tumor. 00:10:21.06 But the red cells that are here, the TGF-beta-responding cells, did not undergo apoptosis. 00:10:28.23 Are they doing something? 00:10:30.03 Perhaps they're just sitting there in the tumor. 00:10:32.09 So, in order to be able to determine this, we washed away the cisplatin, and we activated 00:10:39.07 the lineage-tracing marker so that these cells are now green, and all their progeny are green. 00:10:45.15 If these cells are responsible for regrowing the tumor, the tumor should be green. 00:10:52.00 And so we did that experiment. 00:10:55.06 And what we found is a green tumor. 00:10:57.16 And what this tells us is that by stem cells receiving this change in their microenvironment, 00:11:04.17 that prompts them to receive a TGF-beta signal, causes them to become slower cycling, invasive, 00:11:11.19 and resistant to chemotherapy. 00:11:14.21 So, what's the mechanism involved? 00:11:18.21 And here, there's probably multiple mechanisms. 00:11:21.06 We've looked at one of those. 00:11:23.16 And it involves a factor called NRF2. 00:11:26.17 This is a master regulator of a pathway called the glutathione pathway, and I'll get to that 00:11:32.19 in a moment. 00:11:34.05 NRF2 is normally kept at bay by virtue of its association with an inhibitory protein, 00:11:41.24 KEAP1. 00:11:43.03 And in response to TGF-beta, p21 is upregulated. 00:11:48.04 It competes for the binding of KEAP1 to NRF2. 00:11:53.15 And that then turns NRF2 into a stabilized activator of the glutathione regulatory pathway. 00:12:01.28 The glutathione metabolism genes then go up in the cancer stem cells. 00:12:07.01 And the ones that we identified that were up in the TGF-beta-responding stem cells 00:12:12.00 are those shown in red. 00:12:14.01 So, what's the glutathione pathway doing? 00:12:16.26 The glutathione pathway is what normal, healthy cells use to clear out nasty stuff like cisplatin, 00:12:25.13 in the course of chemotherapy, or reactive oxygen species, in the course of radiotherapy. 00:12:32.26 And so by upregulating the glutathione pathway... normally, it clears out all sorts of 00:12:39.13 nasty stuff from healthy stem cells. 00:12:41.18 But here it's clearing out all sorts of nasty stuff from the cancer stem cells, the cells 00:12:49.01 that we would like to get rid of. 00:12:51.01 So, is this relevant? 00:12:53.04 I've told you about studies in mice -- are they relevant to human? 00:12:56.20 So, here we looked at the glutathione expression program in squamous cell carcinomas of humans. 00:13:04.21 And what we learned is that the glutathione pathway is upregulated in a small cohort of 00:13:10.26 human patients with head and neck squamous cell carcinoma. 00:13:14.18 And when we looked at the prognosis of that small cohort, that cohort with 00:13:19.28 upregulated glutathione pathway genes turns out to be the cohort with the poorest prognosis, 00:13:27.15 suggesting that this is an important finding that could help us in terms of future treatments of cancer. 00:13:34.18 So, NRF2, the good and the bad. 00:13:39.05 NRF2 is what is normally contained in broccoli, in blueberries, and all the things that 00:13:46.15 we're told are really good for our health. 00:13:49.00 They are if we're healthy. 00:13:51.21 But for tumor patients or cancer patients, antioxidants may not necessarily be a good thing. 00:13:58.25 And it's something that is really going to bear the need for future studies, to be able 00:14:03.15 to explore deeper into this. 00:14:05.28 So, how do we go from taking the findings that we've made to, perhaps, the clinics 00:14:13.11 or to new therapies for the treatment of squamous cell carcinoma. 00:14:19.01 Well, to this end, we've engineered isogenic strains, or isogenic cells, that contain 00:14:27.08 the ability... a sensor that allows us to look at whether or not the cells are experiencing 00:14:32.12 TGF-beta or not. 00:14:34.22 We've also engineered the cells such that the... one type of squamous cell carcinoma cell 00:14:41.07 has the TGF-beta receptor on its surface, and we've excised the TGF-beta receptor 00:14:47.21 from the other cell, making it or rendering it unable to respond to TGF-beta. 00:14:53.02 So, here's an example. 00:14:55.11 The red reporter shows us nuclear red if TGF-beta signaling is on. 00:15:00.10 And you can see that if we co-culture the TGF-beta receptor positive and negative cells 00:15:06.00 in a dish, what we find is that when we add TGF-beta the cells that have the receptor 00:15:12.22 show nuclear red -- TGF-beta is on and signaling in those cells -- 00:15:17.15 the green ones, TGF-beta is unable to signal. 00:15:20.25 So, now we can treat the cells with cisplatin. 00:15:24.04 And what we see on the left-hand panel is that if you don't add any TGF-beta and 00:15:31.01 you just add cisplatin, you kill all of the cells, as evidenced by the fact that they round up 00:15:37.01 and die. 00:15:38.06 On the right-hand side, if you treat the cells with TGF-beta, those cells able 00:15:43.10 to respond to TGF-beta are surviving the cisplatin, much the same as what I described to you for the tumor. 00:15:51.25 So, we can also see gamma-H2AX, a sign of DNA damage. 00:15:56.17 And again, DNA damage occurs in the cells that cannot respond to TGF-beta, 00:16:04.09 and the cells that can respond to TGF-beta are refractory. 00:16:07.04 So, what this tells us is that, basically, now we can start to think about therapeutics 00:16:13.19 that would be able to compromise the ability of these slow cycling cancer stem cells 00:16:19.26 that have high levels of glutathione to be able to kill those cells in the presence of, perhaps, 00:16:26.09 combinatorial drugs for... for these cells. 00:16:31.04 We also think that it might be possible, through combinatorial drugs and the use of TGF-beta inhibitors 00:16:39.08 in a very co... in a very specific way, to first kill off the bulk of the tumor cells 00:16:43.28 with cisplatin, for instance, and then mobilize those cancer stem cells 00:16:50.24 that are resistant with, perhaps, TGF-beta inhibitor drugs or antibodies, and then hit the tumor 00:16:57.28 again with cisplatin to wipe out the mobilized, activated cancer stem cells. 00:17:03.11 So, these are ideas that at the moment are just ideas, but we'd like to be able 00:17:08.25 to take these ideas to the practice, and ultimately to translational medicine. 00:17:14.11 But there are many mutations, genetic mutations, that exist within these human squamous cell carcinomas, 00:17:22.24 these solid tumors. 00:17:24.17 And ultimately, we'd like to know which ones are cancer-driving and which ones aren't. 00:17:30.15 So again, my laboratory has said whether we could harness the power of fly and worms, 00:17:37.20 and take advantage of fly and worm genetics, only apply it to the laboratory mouse. 00:17:43.03 Could... to be able to screen through and find out which of the genes that have been 00:17:48.13 mutated in cancer are actually causing the cancer. 00:17:52.17 And so here, two of my former lab members 00:17:57.22 -- Slobodan Beronja, who's running his own laboratory at the Fred Hutchinson Cancer Center, 00:18:02.16 and Geulah Livshits, who was a graduate student and is now postdoccing in a cancer laboratory -- 00:18:10.00 came up with a method of carrying out rapid genetics in mice. 00:18:15.08 What they did was to take advantage of the fact that lentivirus only gets into the 00:18:21.09 very first cell layer that it sees, which, right after gastrulation, is the single layer 00:18:27.09 of surface ectodermal cells that are going to give rise to the epidermis, the hair follicles, 00:18:33.01 the mammary gland, the corneal covering of the eye, and the sweat glands. 00:18:38.15 And what they did was to, then, put the mother mouse under anesthesia at nine and a half days 00:18:45.15 of development, right after gastrulation, with her pups, and basically use ultrasound 00:18:51.19 to find the pups, and then use a microinjection needle to be able to inject high-titer lentivirus 00:18:59.07 just in the amniotic sac, the fluid that the embryo is bathing in. 00:19:03.14 So, in a non-invasive way, we expose the embryo to lentivirus. 00:19:10.09 We now seal up mom, let the pups develop. 00:19:13.09 In this case, we analyze the pups six days later. 00:19:16.11 And what we find is that the lentivirus is now integrated stably into the mouse genome. 00:19:22.27 And it only infected the cells of the epithelium, and did not get into the inner layers beneath it, 00:19:30.26 the dermis. 00:19:32.02 And what we find is that the red embryo, in this case carrying... the lentivirus carried 00:19:38.10 a red fluorescent protein, is now nicely infected over the entire mouse embryo epithelium. 00:19:46.20 And when you look at a cross-section of the tissue, you see that the epidermis and the hair follicles, 00:19:52.01 but no other cells of the skin, basically, were stably transduced with lentivirus. 00:19:59.13 So, what this allows us to do is put any gene we want, or down regulate any gene that we want, 00:20:05.14 or CRISPR/Cas edit any gene that we want, specifically in the skin epithelium 00:20:12.00 in a matter of days. 00:20:13.20 That used to take my laboratory years to accomplish. 00:20:18.03 Ten years later, we've basically been able to accelerate the pace at which we do research 00:20:25.00 in the laboratory. 00:20:26.22 We've now carried out rapid functional genetic screens... genes for... screening for, 00:20:34.17 what are the oncogenes in that group of different human mutations that are found in squamous cell carcinomas? 00:20:41.13 Who are the tumor suppressors? 00:20:44.11 What are the microRNAs that are drivers of squamous cell carcinoma? 00:20:51.18 And what genes are important in regulating the balance of growth and differentiation 00:20:56.18 in the skin epithelium? 00:20:58.23 These technologies and the advances that we've been able to make... carrying out even 00:21:03.27 whole genome-wide screens in mice, which were previously, just a few years ago, thought to be possible 00:21:10.14 only in worms or in fruit flies. 00:21:14.17 So, I hope I've been able to give you the insights, today, of why my laboratory studies the skin, 00:21:21.04 and why we continue to use it as an important model to be able to uncover 00:21:27.23 the basis of genetic disorders, and also the basis of cancers. 00:21:34.04 And for the goal, the broader objective, of basically making advances in the treatments 00:21:41.20 of human genetic diseases. 00:21:44.12 The work that we do is focused on mouse. 00:21:47.25 And there's always that need to be able to draw parallels between mouse and human. 00:21:52.18 But those kinds of approaches are now possible. 00:21:55.27 This is my laboratory. 00:21:57.17 It's an international laboratory of researchers from around the world who get together 00:22:03.20 with a common goal. 00:22:05.07 And that is to work together to be able to advance our knowledge of biology and, ultimately, 00:22:12.20 to make advances that can be useful in the clinics, and in the biotechnology and pharmaceutical industries. 00:22:20.19 Thank you.