1 00:00:00 --> 00:00:04 The following content is provided by MIT OpenCourseWare under a Creative 2 00:00:04 --> 00:00:08 Commons license. Additional information about our 3 00:00:08 --> 00:00:12 license and MIT OpenCourseWare in general is available 4 00:00:12 --> 00:00:20 at ocw.mit.edu 5 00:00:20 --> 00:00:24 We're going to continue our discussion about cancer and cancer 6 00:00:24 --> 00:00:29 genetics today. One small point of clarification: 7 00:00:29 --> 00:00:34 apparently some people were confused about my out of balance scales here 8 00:00:34 --> 00:00:38 related to proliferation and cell death, pointing out that if there 9 00:00:38 --> 00:00:43 were more proliferation and less cell death, then the scale should 10 00:00:43 --> 00:00:48 shift this way instead of this way. That's an astute observation. 11 00:00:48 --> 00:00:53 It doesn't really matter which way the scales point really. 12 00:00:53 --> 00:00:58 The point was that they are out of balance, which is critical for 13 00:00:58 --> 00:01:03 tumorigenesis. But anyway, in review, 14 00:01:03 --> 00:01:08 last time I told you that cancer cells arise from normal cells over a 15 00:01:08 --> 00:01:13 series of events that relate to mutations in cellular genes. 16 00:01:13 --> 00:01:18 And I gave you lots of evidence that things that cause cancer affect our 17 00:01:18 --> 00:01:23 genes, and that our genes are disrupted in cancer cells. 18 00:01:23 --> 00:01:28 And I briefly mentioned that we have evidence for mutations in 19 00:01:28 --> 00:01:34 cellular genes in cancer cells. And I'll show you that again today. 20 00:01:34 --> 00:01:38 But all of that brings us to the point of needing to know, 21 00:01:38 --> 00:01:43 what are the genes? What are the genes that are affected in cancer? 22 00:01:43 --> 00:01:48 And this is important to help us understand how cancers arise, 23 00:01:48 --> 00:01:53 so from a basic biomedical research point of view, 24 00:01:53 --> 00:01:57 but also as we'll see next time, this information is actually very 25 00:01:57 --> 00:02:02 useful in teaching us how to diagnose cancer more effectively or 26 00:02:02 --> 00:02:06 treat it more effectively. And I'll just show this slide, 27 00:02:06 --> 00:02:09 which I showed at the end again as a summary. The cancer cells arise 28 00:02:09 --> 00:02:13 from normal cells through the acquisition of 29 00:02:13 --> 00:02:16 mutations in cellular genes through this process which we call clonal 30 00:02:16 --> 00:02:19 evolution, more and more abnormal cells growing out from this 31 00:02:19 --> 00:02:23 accumulating mass of cells. And as we'll discuss today, 32 00:02:23 --> 00:02:26 these genes affect proliferation and cell death, this out of balance 33 00:02:26 --> 00:02:30 scheme that I alluded to before, but not only these processes. 34 00:02:30 --> 00:02:34 And I emphasize this because these other important processes get a 35 00:02:34 --> 00:02:38 little bit of short shrift in our discussions. But angiogenesis, 36 00:02:38 --> 00:02:42 this process of recruiting a new blood supply in cancer is very 37 00:02:42 --> 00:02:46 important, and also an important therapeutic target. 38 00:02:46 --> 00:02:50 Cell motility, movement of cells, as I mentioned, it's important to 39 00:02:50 --> 00:02:54 get cells away from the initial mass in the process of metastasis, 40 00:02:54 --> 00:02:58 and invasion likewise. And several other processes are also affected in 41 00:02:58 --> 00:03:03 the course of tumorigenesis. So what are the genes? 42 00:03:03 --> 00:03:08 How do we know which genes are affected in cancer? 43 00:03:08 --> 00:03:14 Well, this set of experiments, really, has occurred over the course 44 00:03:14 --> 00:03:19 of arguably the last century. I'm trying to find the genes that 45 00:03:19 --> 00:03:24 are mutated in cancer cells. And many people point to landmark 46 00:03:24 --> 00:03:29 experiments done in 1910 by an investigator at the Rockefeller 47 00:03:29 --> 00:03:35 University by the name of Payton Rauss. 48 00:03:35 --> 00:03:41 Payton Rauss was a virologist, studied viruses at Rockefeller 49 00:03:41 --> 00:03:47 University. And one day, a farmer from Long Island brought a 50 00:03:47 --> 00:03:54 prize-winning hen to this famous doctor at Rockefeller University in 51 00:03:54 --> 00:04:00 New York because the hen was sick. Specifically, the hen had a big 52 00:04:00 --> 00:04:06 tumor in its breast muscle. And if you remember, that would 53 00:04:06 --> 00:04:12 mean the tumor was a sarcoma. And he wanted the doctor to cure his 54 00:04:12 --> 00:04:16 prize-winning hen. Payton Rauss said, 55 00:04:16 --> 00:04:20 thank you, I'll do what I can, and then prominently killed the bird 56 00:04:20 --> 00:04:24 in order to try to study what was giving rise to this tumor. 57 00:04:24 --> 00:04:43 And he did a famous experiment. 58 00:04:43 --> 00:04:47 He took the tumor, ground it up, filtered it so that 59 00:04:47 --> 00:04:51 there were no cells present in the filtrate, and also used a small 60 00:04:51 --> 00:04:55 enough pour filter so that bacteria, which were also known at that time, 61 00:04:55 --> 00:04:59 were removed. So, he had a filtrate that contained no 62 00:04:59 --> 00:05:04 cells, no cancer cells, and no bacteria. 63 00:05:04 --> 00:05:09 And he took this and injected it into a non-tumor bearing bird. 64 00:05:09 --> 00:05:15 And he observed over time that this bird developed a tumor. 65 00:05:15 --> 00:05:21 So he was able to demonstrate the transmissibility of something which 66 00:05:21 --> 00:05:27 could cause cancer in an otherwise unaffected animal. 67 00:05:27 --> 00:05:33 Because it wasn't cells and it wasn't bacteria, 68 00:05:33 --> 00:05:39 he suggested that it was a virus. 69 00:05:39 --> 00:05:43 And it turned out to be, over the course of the next 50 years 70 00:05:43 --> 00:05:48 or so, the virus which was responsible for this disease was 71 00:05:48 --> 00:05:53 identified and given the name Rauss sarcoma virus, 72 00:05:53 --> 00:05:57 named after Payton Rauss. And the structure of this virus was 73 00:05:57 --> 00:06:02 also determined. It was found to be a retrovirus. 74 00:06:02 --> 00:06:07 Retroviruses are in the class of HIV. 75 00:06:07 --> 00:06:12 Their genomes are made of RNA, and they convert it to a DNA form. 76 00:06:12 --> 00:06:17 We'll learn a little bit more about retroviruses in a few lectures. 77 00:06:17 --> 00:06:22 It was a retrovirus, and the structure of its genome was 78 00:06:22 --> 00:06:27 determined eventually. And it was found to contain in its 79 00:06:27 --> 00:06:33 genome the genes that the virus required for replication. 80 00:06:33 --> 00:06:37 And all viruses of this class has a very similar set of replication 81 00:06:37 --> 00:06:42 genes. They need to produce more virus. But at the end of the viral 82 00:06:42 --> 00:06:47 genome, at the extreme right side of the viral genome, 83 00:06:47 --> 00:06:52 there was another gene which was given the name sarc for sarcoma. 84 00:06:52 --> 00:06:57 And it was termed an oncogene, onco for mass, 85 00:06:57 --> 00:07:02 oncology, cancer associated gene, sarc-oncogene. 86 00:07:02 --> 00:07:06 So, the virus it was found can cause cancer in birds by virtue of 87 00:07:06 --> 00:07:11 transferring into those cells a potent cancer associated gene called 88 00:07:11 --> 00:07:15 sarc. And Payton Rauss went onto win the Nobel Prize, 89 00:07:15 --> 00:07:20 actually about 50 years later in the early 1960s for this work on the 90 00:07:20 --> 00:07:24 discovery of Rauss sarcoma virus. And it was extremely important. 91 00:07:24 --> 00:07:29 But it was still a mystery as to where this cancer associated gene 92 00:07:29 --> 00:07:34 came from. Where did the sarc gene originate? Was it a viral gene 93 00:07:34 --> 00:07:38 which the virus used in some fashion to cause cells to proliferate, 94 00:07:38 --> 00:07:43 perhaps for its own replication? Or did it come from some other 95 00:07:43 --> 00:07:48 source, some other origin? And here in the story entered two 96 00:07:48 --> 00:07:54 researchers, Mike Bishop and Harold Varmus. Mike Bishop is now 97 00:07:54 --> 00:07:59 chancellor at UCSF. Harold Varmus is president at 98 00:07:59 --> 00:08:04 Memorial Sloan-Kettering. They did this work at UCSF, 99 00:08:04 --> 00:08:09 and I got quite familiar with it because I ended up working for 100 00:08:09 --> 00:08:14 Harold Varmus as a Ph.D student. This work was done 101 00:08:14 --> 00:08:19 before I got there in about 1975. And they set out to ask this 102 00:08:19 --> 00:08:24 question, what is the origin of this sarc-oncogene? 103 00:08:24 --> 00:08:29 Where did it come from? And what they were able to show was 104 00:08:29 --> 00:08:34 that this Rauss sarcoma virus derived from a virus that was 105 00:08:34 --> 00:08:40 related to it called avian leucosis virus. 106 00:08:40 --> 00:08:46 And avian leucosis virus had only those replication genes, 107 00:08:46 --> 00:08:52 the genes that the virus uses to produce more virus what they were 108 00:08:52 --> 00:08:58 able to show was that this avian leucosis virus, 109 00:08:58 --> 00:09:04 when it infects cells, and here's a chicken cell, 110 00:09:04 --> 00:09:10 and here's the nucleus of that cell, inside that cell are normal copies 111 00:09:10 --> 00:09:16 of the sarc gene. They were able to show that the 112 00:09:16 --> 00:09:20 virus actually acquires this sarc-oncogene from the cells that it 113 00:09:20 --> 00:09:24 infects. Now, it doesn't do that every time. 114 00:09:24 --> 00:09:28 In fact, most of the time, the virus just goes in and replicates 115 00:09:28 --> 00:09:39 and produces more virus. 116 00:09:39 --> 00:09:44 But rarely, and really it's very rarely, the virus actually acquires 117 00:09:44 --> 00:09:49 a piece of the sarc gene and sticks it into its own genome, 118 00:09:49 --> 00:09:56 thereby creating -- 119 00:09:56 --> 00:10:02 -- Rauss sarcoma virus, which has the structure that I 120 00:10:02 --> 00:10:08 showed you above with the replication genes and 121 00:10:08 --> 00:10:14 the sarc oncogene. And this is done through a process 122 00:10:14 --> 00:10:21 called transduction, which is a form of recombination. 123 00:10:21 --> 00:10:28 The main point of this observation, 124 00:10:28 --> 00:10:32 and the really important point of this observation was this potent 125 00:10:32 --> 00:10:36 oncogene is present in chicken cells. 126 00:10:36 --> 00:10:47 The sarc is a cellular gene. 127 00:10:47 --> 00:10:53 It's not just a viral gene. It's derived from host cells of chickens, 128 00:10:53 --> 00:10:59 and it's not only in chickens. It's in all vertebrates including 129 00:10:59 --> 00:11:04 humans. So, you all have a sarc gene in your 130 00:11:04 --> 00:11:09 cells, too. And this was a really momentous observation. 131 00:11:09 --> 00:11:14 And it led Bishop and Varmus to win the Nobel Prize back in 1989. 132 00:11:14 --> 00:11:19 So, what I've just told you is that your cells carry a potent oncogene, 133 00:11:19 --> 00:11:25 sarc. So, why don't you all have sarcomas? 134 00:11:25 --> 00:11:31 Anybody? Remember, 135 00:11:31 --> 00:11:37 we are still being filmed. Anybody? Yes, somebody's got their 136 00:11:37 --> 00:11:42 hand up, but I don't see them. Yes? Almost right. The gene is 137 00:11:42 --> 00:11:48 not expressed properly, OK? The virus has co-opted this 138 00:11:48 --> 00:11:53 gene and placed it into its own genome. And now this gene is being 139 00:11:53 --> 00:11:59 regulated improperly. Maybe it's expressed too well or at 140 00:11:59 --> 00:12:05 the wrong time, or in the wrong cells. 141 00:12:05 --> 00:12:09 And some combination of those things allows the gene to cause 142 00:12:09 --> 00:12:14 cancer in this setting, whereas when the gene is properly 143 00:12:14 --> 00:12:18 regulated, and the cells of chickens or in you, it's not cancer 144 00:12:18 --> 00:12:23 associated. It doesn't cause cancer in its state in the cellular genome. 145 00:12:23 --> 00:12:27 So, this was extremely important, and as I said, 146 00:12:27 --> 00:12:32 led to the Nobel Prize for these guys. RSV -- 147 00:12:32 --> 00:12:46 -- is what's called an acutely 148 00:12:46 --> 00:12:50 transforming retrovirus, acutely transforming meaning that it 149 00:12:50 --> 00:12:54 can transform cells, turn them from normal to cancerous 150 00:12:54 --> 00:12:59 quickly. And as I said, it's a retrovirus. We now know of 151 00:12:59 --> 00:13:03 many acutely transforming viruses of animals: mice, 152 00:13:03 --> 00:13:08 rats, chickens, turkeys, other things. 153 00:13:08 --> 00:13:13 And from them, we have learned the identity of 154 00:13:13 --> 00:13:18 about 50 or so oncogenes, all of which were derived from the 155 00:13:18 --> 00:13:23 host cell. So, we know from this type of experiment 156 00:13:23 --> 00:13:28 of many potential cancer associated genes. Importantly, 157 00:13:28 --> 00:13:34 there are no known acutely transforming viruses of humans. 158 00:13:34 --> 00:13:39 Importantly, the vast majority of human cancers are not associated 159 00:13:39 --> 00:13:44 with viral agents. Occasionally, there are such cases, 160 00:13:44 --> 00:13:49 but they are quite rare. Most major tumor types are not caused by viral 161 00:13:49 --> 00:13:55 infection. But there is one big exception to this -- 162 00:13:55 --> 00:14:04 -- human papilloma virus. 163 00:14:04 --> 00:14:10 Human papilloma virus, or HPV, and specifically high-risk types of 164 00:14:10 --> 00:14:16 HPV called type 16 and type 18 are associated with cervical cancer in 165 00:14:16 --> 00:14:21 the US, but mostly in other parts of the world. And that's important, 166 00:14:21 --> 00:14:27 because if one can control exposure to HPV, or eradication of HPV, 167 00:14:27 --> 00:14:33 one could do something about cervical cancer, 168 00:14:33 --> 00:14:38 and that's actually happening now. And I'll mention that again next 169 00:14:38 --> 00:14:42 lecture. But I want you to remember that most cancers, 170 00:14:42 --> 00:14:46 human cancers, are not virus associated. But the viruses have 171 00:14:46 --> 00:14:50 taught us a great deal about cancer genetics. OK, 172 00:14:50 --> 00:14:54 so if most human cancers don't arise from a viral infection, 173 00:14:54 --> 00:14:58 the infection with an oncogene carrying virus, 174 00:14:58 --> 00:15:03 then where do these mutations come from? 175 00:15:03 --> 00:15:08 What are the mutations in human cancer? And here, 176 00:15:08 --> 00:15:13 just after these experiments were done, around 1980, 177 00:15:13 --> 00:15:18 comes another one of our local heroes, Bob Weinberg, 178 00:15:18 --> 00:15:23 who has a laboratory at the Whitehead Institute here at MIT. 179 00:15:23 --> 00:15:28 He hasn't yet won the Nobel Prize, but I think he's going to, based on 180 00:15:28 --> 00:15:34 this work and a lot of other work probably in the next several years. 181 00:15:34 --> 00:15:40 And what Weinberg's lab did back in this early 1980s. 182 00:15:40 --> 00:15:47 Was to look in human cancers, so not cancers of animals, but in 183 00:15:47 --> 00:15:54 human cancers. And specifically, 184 00:15:54 --> 00:16:01 they took bladder cancers and isolated the cells, 185 00:16:01 --> 00:16:13 and grew them in the laboratory. 186 00:16:13 --> 00:16:15 They assumed that inside of the cells were altered genes, 187 00:16:15 --> 00:16:17 and to become altered through the life of this individual, 188 00:16:17 --> 00:16:20 giving rise to this cancer. And they wanted to know what those 189 00:16:20 --> 00:16:22 genes were. And so, they isolated the DNA from these 190 00:16:22 --> 00:16:25 bladder cancer cells, and they transfected it into cells 191 00:16:25 --> 00:16:27 from mice. They sheared it up, and they introduced it. That's what 192 00:16:27 --> 00:16:30 transfected means, into the cells of mice. 193 00:16:30 --> 00:17:05 And these cells that they put them into were fairly normal -- 194 00:17:05 --> 00:17:13 -- mouse cells, 195 00:17:13 --> 00:17:17 by which I mean they grew a single layer on the bottom of the dish. 196 00:17:17 --> 00:17:21 There are structures look like normal cells as opposed to cancer 197 00:17:21 --> 00:17:25 cells. They were relatively normal cells. And what they observed was 198 00:17:25 --> 00:17:29 that a small percentage of these cells that had picked up some bits 199 00:17:29 --> 00:17:33 and pieces of the DNA derived from this human cancer began to 200 00:17:33 --> 00:17:41 behave abnormally -- 201 00:17:41 --> 00:17:47 -- and produced what were referred to as transformed cells, 202 00:17:47 --> 00:17:53 cells that looked more like cancer cells. So they had converted 203 00:17:53 --> 00:17:59 otherwise normal mouse cells to what looked like cancer cells through the 204 00:17:59 --> 00:18:06 introduction of one or more human genes. 205 00:18:06 --> 00:18:13 And they further demonstrated that these were cancer associated cells, 206 00:18:13 --> 00:18:20 cancer-causing cells, by introducing them into immunocompromised mice, 207 00:18:20 --> 00:18:28 and observing that over time these cells could produce tumors. 208 00:18:28 --> 00:18:34 So these were cancer cells indeed. So that's very interesting because 209 00:18:34 --> 00:18:39 it says that there are altered genes in these cancer cells that can 210 00:18:39 --> 00:18:45 convert normal mouse cells into cancer cells. And then to obvious 211 00:18:45 --> 00:18:50 question was, what are those genes? What is the gene that's responsible 212 00:18:50 --> 00:19:08 for this process? So, they -- 213 00:19:08 --> 00:19:12 -- isolated the human DNA from the transformed mouse cells. 214 00:19:12 --> 00:19:17 And I won't tell you how they did that, but suffice it to say, 215 00:19:17 --> 00:19:22 it's possible to isolate the human DNA away from the mouse DNA in these 216 00:19:22 --> 00:19:26 transformed mouse cells. They cloned the human DNA into 217 00:19:26 --> 00:19:31 bacteria using recombinant DNA methods that we've talked to you 218 00:19:31 --> 00:19:44 about previously. 219 00:19:44 --> 00:19:46 And they sequenced the cancer-causing gene. 220 00:19:46 --> 00:19:48 It turns out it was just a single gene that was providing 221 00:19:48 --> 00:19:55 this property. 222 00:19:55 --> 00:19:59 And it turned out that this gene was H-RAS, RAS being a gene that 223 00:19:59 --> 00:20:03 we've talked to you about before, and important signaling protein in 224 00:20:03 --> 00:20:07 cells. Moreover, they sequenced the copy of RAS that 225 00:20:07 --> 00:20:11 was present in the cancer cells, and compared it to the sequence in 226 00:20:11 --> 00:20:15 normal cells in the other cells of this individual. 227 00:20:15 --> 00:20:20 And they found a single alteration. OK, so what you are looking at here 228 00:20:20 --> 00:20:24 now is the sequence of the H-RAS gene in normal people or in the 229 00:20:24 --> 00:20:28 normal cells of this cancer patient has a particular sequence. 230 00:20:28 --> 00:20:32 It encodes a protein, H-RAS, which if you recall is an important 231 00:20:32 --> 00:20:36 signaling protein that shuttles between a GTP-bound form 232 00:20:36 --> 00:20:41 and a GDP bound form. In its GTP bound form, 233 00:20:41 --> 00:20:45 it's active and signals, and then it undergoes hydrolysis 234 00:20:45 --> 00:20:49 reaction. So, the GTP is converted to GDP, 235 00:20:49 --> 00:20:53 and becomes inactive. The mutant copy found in the cancer cells and 236 00:20:53 --> 00:20:57 in these transformed mouse cells has been alteration which blocks the 237 00:20:57 --> 00:21:01 ability of the proteins undergo GTP hydrolysis. Therefore, 238 00:21:01 --> 00:21:05 the protein gets locked into this active signaling state, 239 00:21:05 --> 00:21:09 and stimulates proliferation continuously. 240 00:21:09 --> 00:21:14 And that's why it's a cancer associated gene. 241 00:21:14 --> 00:21:19 OK, so here we have for the first time evidence that a normal, 242 00:21:19 --> 00:21:24 cellular gene gets mutated, presumably by an error or by 243 00:21:24 --> 00:21:29 exposure to some mutagen that is responsible at least far an aspect 244 00:21:29 --> 00:21:34 of the transformed state. OK, so that's very, 245 00:21:34 --> 00:21:40 very interesting. It also raises an important question. 246 00:21:40 --> 00:21:45 If you note that the change between the normal gene and the abnormal 247 00:21:45 --> 00:21:51 gene is signal-base change that converts this alanine residue, 248 00:21:51 --> 00:21:56 sorry, this glycemic residue to a valine residue: single base change. 249 00:21:56 --> 00:22:02 Such single base changes occur in your cells all the time. 250 00:22:02 --> 00:22:07 It's estimated that the mutation rate inside your cells is about ten 251 00:22:07 --> 00:22:12 to the minus ninth per cell division. And if you calculate how many cells 252 00:22:12 --> 00:22:17 you have in your body at any given time based on how many cell 253 00:22:17 --> 00:22:22 divisions have arisen, we can estimate that there are about 254 00:22:22 --> 00:22:28 ten to the third to ten to the fifth -- 255 00:22:28 --> 00:22:35 -- rasmutin 256 00:22:35 --> 00:22:41 cells in all of U as you sit there now. You are all carrying 1, 257 00:22:41 --> 00:22:46 00-100,000 cells that have this very mutation. So, 258 00:22:46 --> 00:22:52 why don't all of you have cancer? Why don't all of you have bladder 259 00:22:52 --> 00:22:58 cancer or some other form of cancer? Anybody? 260 00:22:58 --> 00:23:05 The reason is that cancer is not a 261 00:23:05 --> 00:23:09 single step process. Cancer cells arise from normal 262 00:23:09 --> 00:23:13 cells to the acquisition of many mutations that occur over the 263 00:23:13 --> 00:23:17 lifetime of the individual. So a single mutation in RAS is not 264 00:23:17 --> 00:23:21 enough to give you a cancer. It might be enough to cause the 265 00:23:21 --> 00:23:25 cells to behave somewhat abnormally. But that in and of itself does not 266 00:23:25 --> 00:23:29 produce even hyperplasia necessarily, let alone an adenoma, 267 00:23:29 --> 00:23:34 an adenocarcinoma and invasive cancer. 268 00:23:34 --> 00:23:37 It's necessary to acquire multiple mutations. So you may have cells 269 00:23:37 --> 00:23:41 that are on their way. But they are not fully there. 270 00:23:41 --> 00:23:45 And hopefully they'll never get there. Now, I want to remind you 271 00:23:45 --> 00:23:49 that this pathway, this signaling pathway that involves 272 00:23:49 --> 00:23:53 RAS is something we taught you about previously. This should look 273 00:23:53 --> 00:23:57 familiar based on the cell signaling lectures that we had before. 274 00:23:57 --> 00:24:01 RAS sits right here, this important signaling molecule that links events 275 00:24:01 --> 00:24:05 that take place at the cell membrane. 276 00:24:05 --> 00:24:09 Sound at all familiar? Yes? Transmembrane receptors? 277 00:24:09 --> 00:24:13 Growth factor receptors that bind to ligands and cause a series of 278 00:24:13 --> 00:24:17 events that for example convert RAS to its GTP bound form, 279 00:24:17 --> 00:24:22 and then initiate a kinase cascade of protein kinases, 280 00:24:22 --> 00:24:26 map kinase, kinase, kinase, kinase, kinase, kinase, remember the 281 00:24:26 --> 00:24:30 little movie? Moves into the nucleus, phosphorylates 282 00:24:30 --> 00:24:34 transcription factors that initiate the expression of target genes that, 283 00:24:34 --> 00:24:39 for example cause cells to proliferate. 284 00:24:39 --> 00:24:43 And when RAS gets stuck in its on state as it does here, 285 00:24:43 --> 00:24:47 these signals are sent constitutively so that the cell is 286 00:24:47 --> 00:24:51 being told to divide all the time instead of in a regulated fashion. 287 00:24:51 --> 00:24:55 Now, I point out all the different components which I've told you about 288 00:24:55 --> 00:24:59 in previous lectures because, in fact, it's not just RAS, this one 289 00:24:59 --> 00:25:04 signaling protein that gets mutated in cancers. 290 00:25:04 --> 00:25:08 Many different of these signaling proteins get mutated in cancers. 291 00:25:08 --> 00:25:12 It is a very common pathway that's affected in not necessarily all but 292 00:25:12 --> 00:25:16 a very high percentage of cancers of different types. 293 00:25:16 --> 00:25:20 The RAS gene is mutated at about 30% of cancers. 294 00:25:20 --> 00:25:24 30% of all cancers have mutations in one of the RAS genes including, 295 00:25:24 --> 00:25:28 for example, 90% of pancreatic cancer, 50% of colon cancers, 296 00:25:28 --> 00:25:33 30% of lung cancers carry mutations in this gene. 297 00:25:33 --> 00:25:36 But other components get mutated as well. For example, 298 00:25:36 --> 00:25:40 the growth factor or growth factor receptor genes get altered in 299 00:25:40 --> 00:25:43 cancers. They become amplified. And I'll mention what I mean by 300 00:25:43 --> 00:25:47 that in a second period. We observe translocations, 301 00:25:47 --> 00:25:50 these chromosomal alterations in different genes, 302 00:25:50 --> 00:25:54 including the transcription factor genes that lie at the bottom of this 303 00:25:54 --> 00:25:57 pathway. There can be deletions that affect the function of these 304 00:25:57 --> 00:26:01 proteins, make the function abnormally, and subtle mutations 305 00:26:01 --> 00:26:04 like the one in RAS itself. And there are other examples, 306 00:26:04 --> 00:26:08 subtle mutations that lock the proteins, for example, 307 00:26:08 --> 00:26:21 in an active signaling state. 308 00:26:21 --> 00:26:27 So, DNA amplification is one such mechanism to alter the function of a 309 00:26:27 --> 00:26:33 cellular gene in cancer. And an example of that is in a case 310 00:26:33 --> 00:26:45 of a gene called HER2. 311 00:26:45 --> 00:26:51 It encodes a growth factor receptor like item number two up here. 312 00:26:51 --> 00:26:58 It encodes a growth factor receptor. And when it's present in single 313 00:26:58 --> 00:27:04 copy or two copies per cell, it produces a certain concentration 314 00:27:04 --> 00:27:11 of this growth factor receptor protein. And it signals 315 00:27:11 --> 00:27:17 at normal levels. And that gives rise to a normal 316 00:27:17 --> 00:27:21 amount of proliferation. And this is particularly important 317 00:27:21 --> 00:27:25 in cells of the mammary gland. Mammary epithelial cells depend on 318 00:27:25 --> 00:27:30 this signaling pathway to be produced in the right amounts. 319 00:27:30 --> 00:27:36 Unfortunately at some frequency in cancer cells, and specifically 320 00:27:36 --> 00:27:42 cancer cells of the breast, and sometimes ovary, an 321 00:27:42 --> 00:27:48 amplification event takes place so that now instead of having just one 322 00:27:48 --> 00:27:54 copy of this HER2 gene on the chromosome, there are many copies. 323 00:27:54 --> 00:28:00 An error takes place in the replication process so that instead 324 00:28:00 --> 00:28:06 of just copying this gene one time, it gets copied multiple times. 325 00:28:06 --> 00:28:12 So, now we have too many copies of this gene, and therefore in these 326 00:28:12 --> 00:28:19 cells, the concentration of this receptor is significantly higher 327 00:28:19 --> 00:28:26 than in normal cells such that in these cells, there might be ten 328 00:28:26 --> 00:28:33 times or a hundred times the amount of signaling, or ten to a hundred 329 00:28:33 --> 00:28:38 times the amount of proliferation. So, the dysregulation of this gene's 330 00:28:38 --> 00:28:42 function by making too much of the protein, it's an otherwise normal 331 00:28:42 --> 00:28:46 protein. There's just too much of it, causes the cells to get 332 00:28:46 --> 00:28:50 overstimulated and divide too often. That's bad news for cancer. The 333 00:28:50 --> 00:28:54 good news is we now have a therapy, and I'll put you about that next 334 00:28:54 --> 00:28:58 time, directed against this protein. 335 00:28:58 --> 00:29:07 I'll mention another mechanism of 336 00:29:07 --> 00:29:11 activation, and that's translocation. I showed you slides of cancer cells 337 00:29:11 --> 00:29:16 that have broken chromosomes or rearranged chromosomes. 338 00:29:16 --> 00:29:20 Those rearrangements often produce abnormal genes, 339 00:29:20 --> 00:29:25 sometimes fusions between one gene and another. Other times, 340 00:29:25 --> 00:29:29 rearrangements of the promoter of the gene such that the normal 341 00:29:29 --> 00:29:34 promoter of the gene, which is responsible for its level 342 00:29:34 --> 00:29:38 of expression is replaced by a different promoter, 343 00:29:38 --> 00:29:43 which might give too much expression. And that's true for an important 344 00:29:43 --> 00:29:48 oncogene called MIC. The MIC oncogene is a 345 00:29:48 --> 00:29:53 transcription factor. It's like number eight down there. 346 00:29:53 --> 00:29:58 It's a transcription factor that binds to DNA and regulates the 347 00:29:58 --> 00:30:03 expression of other genes. And it's normally expressed from a, 348 00:30:03 --> 00:30:09 let's call it, weak promoter. You don't want to have too much of this 349 00:30:09 --> 00:30:15 protein produced. It needs to be produced at normal 350 00:30:15 --> 00:30:21 levels. So, we get a certain concentration of the 351 00:30:21 --> 00:30:32 protein, which gives -- 352 00:30:32 --> 00:30:37 -- regulated cell division. You get as much television as you 353 00:30:37 --> 00:30:42 need based on the appropriate signals in the environment of those 354 00:30:42 --> 00:30:47 cells. In certain types of leukemia, one sees a translocation. 355 00:30:47 --> 00:30:52 The DNA is broken and rejoined so that the MIC oncogene doesn't have 356 00:30:52 --> 00:30:57 its own promoter anymore. Instead, it's next to a very strong 357 00:30:57 --> 00:31:04 promoter. 358 00:31:04 --> 00:31:09 And now, again, the concentration of this protein is 359 00:31:09 --> 00:31:15 increased. And this gives rise to deregulated cell 360 00:31:15 --> 00:31:21 division, same idea. Change the proper regulation of 361 00:31:21 --> 00:31:27 this signaling network, and now the signaling network is 362 00:31:27 --> 00:31:31 broken. And as I said, 363 00:31:31 --> 00:31:35 there are many, many such genes that are known. 364 00:31:35 --> 00:31:39 We know about 50 or so oncogenes from these acutely transforming 365 00:31:39 --> 00:31:42 viruses. We probably know of 50 more oncogenes because of their 366 00:31:42 --> 00:31:46 changes in the DNA of cancer cells. And by the way, there's a lot of 367 00:31:46 --> 00:31:50 overlap between these two sets. Many of the genes that were found 368 00:31:50 --> 00:31:53 in the context of viruses were later found to be mutant in human cells 369 00:31:53 --> 00:31:57 having nothing to do with viruses. And RAS is one such example. MIC, 370 00:31:57 --> 00:32:01 actually, is another example, and a relative of this HER2 gene 371 00:32:01 --> 00:32:08 is another example. 372 00:32:08 --> 00:32:16 OK. 373 00:32:16 --> 00:32:21 OK, so the slide is meant to reinforce this notion that cell 374 00:32:21 --> 00:32:27 division, normal production of cells, is tightly regulated through these 375 00:32:27 --> 00:32:32 signaling networks, and that oncogenes function normally, 376 00:32:32 --> 00:32:38 their normal cellular function is to control this process. 377 00:32:38 --> 00:32:42 That's what they do in normal development. That's what they do in 378 00:32:42 --> 00:32:47 normal individuals. But they can be subverted in cancer 379 00:32:47 --> 00:32:51 cells by mutation. It makes them act in a deregulated 380 00:32:51 --> 00:32:56 or uncontrolled fashion. There is another set of genes that 381 00:32:56 --> 00:33:01 act, in a sense, in opposition to the oncogenes. 382 00:33:01 --> 00:33:05 These genes function to inhibit this process of cell division. 383 00:33:05 --> 00:33:09 And they go by another name: tumor suppressor genes. 384 00:33:09 --> 00:33:13 Tumor suppressor genes function to block the normal cell division 385 00:33:13 --> 00:33:18 process or to inhibit the production of cells. And they, 386 00:33:18 --> 00:33:22 too, are important in cancer. You can think of these genes as the 387 00:33:22 --> 00:33:26 on switch and the off switch of an electrical circuit. 388 00:33:26 --> 00:33:31 In cancer cells, oncogenes become hyperactive. 389 00:33:31 --> 00:33:36 When the lights switch gets stuck in the on state. OK, 390 00:33:36 --> 00:33:41 it gets taped open. So now, you're getting signaling 391 00:33:41 --> 00:33:46 through this circuit continuously. Tumor suppressor genes normally 392 00:33:46 --> 00:33:51 inhibit cell division. And so, in cancer, these genes get 393 00:33:51 --> 00:33:56 inactivated. It's like the light switch getting turned off. 394 00:33:56 --> 00:34:01 And the process is normally regulated that way, and 395 00:34:01 --> 00:34:06 these get stuck off. OK, so want to tell you a little bit 396 00:34:06 --> 00:34:10 about tumor suppressor genes now which is the other major category of 397 00:34:10 --> 00:34:14 cancer associated genes that we'll cover here. Briefly, 398 00:34:14 --> 00:34:18 just to reiterate that point, in addition to the light switch 399 00:34:18 --> 00:34:23 analogy, people often use the gas pedal and breaks analogy, 400 00:34:23 --> 00:34:27 or the go signal and stop signal analogy. So, I'll use that as well. 401 00:34:27 --> 00:34:31 In normal cells, when they receive the signals to divide, 402 00:34:31 --> 00:34:35 they are stimulated to go, that is, to divide to make more such 403 00:34:35 --> 00:34:40 cells. This happens in development when you 404 00:34:40 --> 00:34:44 need to make more cells. It happens in wound healing. 405 00:34:44 --> 00:34:48 It happens in normal homeostasis. And then, when the process is 406 00:34:48 --> 00:34:53 completed, there are stop signals sent. The go signals represent 407 00:34:53 --> 00:34:57 oncogenes, normally regulated, stop signals tumor suppressor genes 408 00:34:57 --> 00:35:02 normally regulated. In cancer, oncogenes become 409 00:35:02 --> 00:35:06 deregulated. And pushed too strong they send constitutive signals. 410 00:35:06 --> 00:35:11 And as a consequence, too many cells are produced. 411 00:35:11 --> 00:35:15 And likewise, the stop signs, the stop signals are lost so that 412 00:35:15 --> 00:35:20 one produces even more cells. OK, so hopefully these are useful 413 00:35:20 --> 00:35:24 analogies to think about these two opposing sets of oncogenes and tumor 414 00:35:24 --> 00:35:29 suppressor genes. OK, so to get into tumor suppressor 415 00:35:29 --> 00:35:33 genes, sorry, this just makes the point, oncogene mutations, 416 00:35:33 --> 00:35:38 tumor suppressor gene mutations. To get into tumor suppressor genes, 417 00:35:38 --> 00:35:42 the second class, an important class of human cancer genes, 418 00:35:42 --> 00:35:46 let me tell you a little bit about this disease which is called 419 00:35:46 --> 00:35:50 retinoblastoma. Retinoblastoma is a childhood tumor 420 00:35:50 --> 00:35:54 of the eye, of the retina. It's not very common. Only about 421 00:35:54 --> 00:35:58 one in 40,000 children develop this tumor. But it's very important what 422 00:35:58 --> 00:36:03 it's taught us about cancer genes. And you can see in the normal eye a 423 00:36:03 --> 00:36:09 normal looking retina, and in the cancer containing eye, 424 00:36:09 --> 00:36:15 these blobs of tumors. We now know the gene that's responsible for this 425 00:36:15 --> 00:36:21 disease, the gene that's mutated in the formation of this disease. 426 00:36:21 --> 00:36:28 And that gene is the gene called RB, named after retinoblastoma. 427 00:36:28 --> 00:36:44 And this gene functions as an 428 00:36:44 --> 00:36:51 important regulator of cell cycle progression. If you remember cell 429 00:36:51 --> 00:36:58 cycle progression, mitosis, S phase mitosis, 430 00:36:58 --> 00:37:05 the G1 phase, the G2 phase, again, dim memories from earlier in 431 00:37:05 --> 00:37:11 the class. The RB protein, 432 00:37:11 --> 00:37:16 which goes by the name of PRB, this is the protein encoded by this 433 00:37:16 --> 00:37:22 tumor suppressor gene, RB, acts to inhibit cell cycle 434 00:37:22 --> 00:37:27 progression. That's its function. It blocks cell cycle progression 435 00:37:27 --> 00:37:32 literally like the brakes on a car. Now, you might ask, 436 00:37:32 --> 00:37:37 if it's there and functioning, then how do you ever get cell cycle 437 00:37:37 --> 00:37:43 progression? And the answer is that this active inhibitor can be 438 00:37:43 --> 00:37:48 inactivated through phosphorylation. When this protein gets 439 00:37:48 --> 00:37:54 phosphorylated, it becomes inactive. 440 00:37:54 --> 00:38:00 And now, cell cycle progression 441 00:38:00 --> 00:38:14 can occur. 442 00:38:14 --> 00:38:19 It becomes inactive when the cell receives growth stimulatory signals 443 00:38:19 --> 00:38:24 like through that signaling network that I mentioned previously. 444 00:38:24 --> 00:38:29 These signals, then, act on cycline CDK complexes, 445 00:38:29 --> 00:38:34 cell cycle associated kinases that you were taught about in the cell 446 00:38:34 --> 00:38:39 cycle lecture, and these go about inactivating the 447 00:38:39 --> 00:38:44 RB protein, allowing the cells to cycle. 448 00:38:44 --> 00:38:50 In addition, when the cells shouldn't be cycling in the presence 449 00:38:50 --> 00:38:56 of growth inhibitory signals, inhibitory signals are sent to the 450 00:38:56 --> 00:39:02 cycline dependent kinase proteins, and they don't function, thereby 451 00:39:02 --> 00:39:08 blocking the inactivation of RB. RB stays in its active state and 452 00:39:08 --> 00:39:13 blocks cell cycle progression. OK, so that's how this really 453 00:39:13 --> 00:39:19 important cell cycle regulator functions more or less. 454 00:39:19 --> 00:39:24 And it's a very important human tumor suppressor gene, 455 00:39:24 --> 00:39:30 which is itself mutated and probably 30 or 40% of human cancers. 456 00:39:30 --> 00:39:36 And the pathway that regulates this gene, this pathway, 457 00:39:36 --> 00:39:43 is mutated in probably 95% of human cancers very, very commonly affected. 458 00:39:43 --> 00:39:49 OK, so given what I've told you so far, would you expect the mutations 459 00:39:49 --> 00:39:56 that we find in the RB gene in human cancer to be activating mutations or 460 00:39:56 --> 00:40:01 inactivating mutations? Inactivating. It's the brakes. 461 00:40:01 --> 00:40:04 Cancer cells want to get rid of the brakes. 462 00:40:04 --> 00:40:15 And so we find inactivating 463 00:40:15 --> 00:40:23 mutations in the RB gene in human cancers like deletions or nonsense 464 00:40:23 --> 00:40:31 mutations, that is, stop codon mutations. 465 00:40:31 --> 00:40:35 OK, that's the kind of mutations we find in this gene. 466 00:40:35 --> 00:40:40 For the oncogenes, which we focused on at the beginning, 467 00:40:40 --> 00:40:45 are the mutations in oncogenes dominant or recessive? 468 00:40:45 --> 00:40:49 Are the mutations in the oncogenes dominant or recessive? 469 00:40:49 --> 00:40:54 The oncogene mutations are dominant. When you acquire mutation in an 470 00:40:54 --> 00:40:59 oncogene, whether it's a subtle mutation or an amplification, 471 00:40:59 --> 00:41:04 it doesn't matter what's happening to the other allele. 472 00:41:04 --> 00:41:08 This thing has new function, gain of function. It will transform 473 00:41:08 --> 00:41:12 or start the process of transformation, 474 00:41:12 --> 00:41:16 regardless of what is happening to the other allele. 475 00:41:16 --> 00:41:21 And that's evident, by the way, in this transfection 476 00:41:21 --> 00:41:25 experiment. This would only work if it were a dominant mutation. 477 00:41:25 --> 00:41:29 So, oncogene mutations are dominant mutations. Are tumor suppressor 478 00:41:29 --> 00:41:34 gene mutations dominant or recessive? These are recessive mutations. 479 00:41:34 --> 00:41:38 Here, it matters, the state of the other allele of the 480 00:41:38 --> 00:41:43 RB gene. As long as you have one functional copy of the brakes, 481 00:41:43 --> 00:41:48 you can do this. You can control cell cycle regulation. 482 00:41:48 --> 00:41:52 In order to make this dysregulated, you need to get rid of both copies. 483 00:41:52 --> 00:41:57 You need to get rid of the brakes entirely. So, these mutations 484 00:41:57 --> 00:42:02 are recessive. And this class of mutations are 485 00:42:02 --> 00:42:08 recessive. Tumor suppressor gene mutations are recessive. 486 00:42:08 --> 00:42:14 So this raises an interesting question. Tumor suppressor gene 487 00:42:14 --> 00:42:19 mutations are recessive. I've told you that tumor suppressor 488 00:42:19 --> 00:42:25 gene mutations occur commonly in human cancer, not just this RB gene 489 00:42:25 --> 00:42:31 but others too, and yet you are all diploid. 490 00:42:31 --> 00:42:36 You all carry two copies of the tumor suppressor genes. 491 00:42:36 --> 00:42:41 So, how do we ever find mutations in these genes in human cancer? 492 00:42:41 --> 00:42:46 Is it enough to mutate just one copy? No, because they are 493 00:42:46 --> 00:42:52 recessive mutations. So, to find mutations in tumor 494 00:42:52 --> 00:42:57 suppressor genes in human cancer, it's not enough to mutate just one 495 00:42:57 --> 00:43:03 copy. Both copies need to be mutated. 496 00:43:03 --> 00:43:13 Recessive mutations require there be 497 00:43:13 --> 00:43:18 two mutations. And this is what typically happens 498 00:43:18 --> 00:43:23 in the development of a tumor with a mutation in the RB gene, 499 00:43:23 --> 00:43:27 or any other tumor suppressor gene. Again, there are probably 20 to 50 500 00:43:27 --> 00:43:32 different tumor suppressor genes. Initially, a cell, which has a 501 00:43:32 --> 00:43:37 normal copy of RB on both copies of chromosome 13, 502 00:43:37 --> 00:43:42 that's where this gene sits, and here we are looking at a linked 503 00:43:42 --> 00:43:47 polymorphic marker, big A little a, if you remember 504 00:43:47 --> 00:43:52 polymorphic markers, big A little A. 505 00:43:52 --> 00:44:00 First, one copy of RB gets mutated. In this cell, there is still a 506 00:44:00 --> 00:44:06 normal copy of RB. And therefore, 507 00:44:06 --> 00:44:10 this cell that has acquired this mutation is normal. 508 00:44:10 --> 00:44:14 It's functionally normal. However, it now has just one copy 509 00:44:14 --> 00:44:18 of the RB gene. And so, it's predisposed to 510 00:44:18 --> 00:44:22 becoming a cancer cell if that normal copy gets lost. 511 00:44:22 --> 00:44:26 And what this shows you are various mechanisms by which the 512 00:44:26 --> 00:44:31 normal copy of the RB gene can be lost. 513 00:44:31 --> 00:44:36 The other copy can acquire its own mutation. Or, 514 00:44:36 --> 00:44:42 the chromosome that carries the normal copy of the RB gene can get 515 00:44:42 --> 00:44:47 lost in the developing cancer cell. Or, it can be a recombination of 516 00:44:47 --> 00:44:53 that, that replaces the normal copy of the gene with the abnormal copy 517 00:44:53 --> 00:44:59 of the gene. And all three of these things happen in the development of 518 00:44:59 --> 00:45:05 cancer cells. And depending on which mechanism is used -- 519 00:45:05 --> 00:45:22 -- one can observe something 520 00:45:22 --> 00:45:30 referred to as loss -- 521 00:45:30 --> 00:45:34 -- of heterozygosity, loss of heterozygosity within the 522 00:45:34 --> 00:45:39 cancer cell. You will notice that this cancer cell, 523 00:45:39 --> 00:45:44 sorry, this normal cell, is heterozygous for this A allele, 524 00:45:44 --> 00:45:48 big A little A. And you will notice that here and here, 525 00:45:48 --> 00:45:53 the cell has become, the cell carries only one version of that A 526 00:45:53 --> 00:45:58 allele, big A. So, there was heterozygosity. 527 00:45:58 --> 00:46:03 And in the cancer cell, there's now loss of heterozygosity. 528 00:46:03 --> 00:46:09 Loss of heterozygosity is a hallmark for tumor suppressor genes, 529 00:46:09 --> 00:46:16 the involvement of tumor suppressor genes in the development of cancer. 530 00:46:16 --> 00:46:22 OK, now I told you several times that proliferation is important and 531 00:46:22 --> 00:46:33 cell death is important, too. 532 00:46:33 --> 00:46:38 Cell death control in cancer: I don't have a lot of time to tell you 533 00:46:38 --> 00:46:43 more about this in this class, but I want to just emphasize that 534 00:46:43 --> 00:46:48 the pathways that lead from cell death signals, 535 00:46:48 --> 00:46:53 the signals that tell cells to commit suicide to ultimately undergo 536 00:46:53 --> 00:46:59 this process called apoptosis are also dysregulated in cancer. 537 00:46:59 --> 00:47:07 For example, there's a gene called 538 00:47:07 --> 00:47:13 BCL2, which becomes mutated in certain types of cancer. 539 00:47:13 --> 00:47:20 BCL2 normally blocks this process of cell death. 540 00:47:20 --> 00:47:26 And in this type of cancer, it becomes hyperactive. So there's 541 00:47:26 --> 00:47:33 not as much cell death. BCL2 is an oncogene. 542 00:47:33 --> 00:47:41 Another gene, very important gene in human cancer is P53. 543 00:47:41 --> 00:47:49 It stimulates cell death. P53 gets lost in a high percentage 544 00:47:49 --> 00:47:57 of cancers. It's a tumor suppressor gene, and therefore gets 545 00:47:57 --> 00:48:03 inactivated. BCL2 is an oncogene. 546 00:48:03 --> 00:48:08 It gets hyperactivated in cancers. I'll talk more about this pathway 547 00:48:08 --> 00:48:14 next time, but just to put in your minds, P53 is a very important 548 00:48:14 --> 00:48:19 signaling protein responsive to many signals upstream, 549 00:48:19 --> 00:48:24 giving rise to signals that lead to death as well as arrest. 550 00:48:24 --> 00:48:30 Hang on, please. Hang on. Two minutes left. 551 00:48:30 --> 00:48:34 And this slide is just a summary of what we've been talking about so far. 552 00:48:34 --> 00:48:38 Control of proliferation, control of cell death, the normal 553 00:48:38 --> 00:48:42 balance, perturbations in these pathways, oncogene mutations that 554 00:48:42 --> 00:48:46 stimulate proliferation, tumor suppressor gene mutations that 555 00:48:46 --> 00:48:50 stimulate proliferation. Alterations in apoptosis, 556 00:48:50 --> 00:48:54 oncogene mutations that block apoptosis, tumor suppressor gene 557 00:48:54 --> 00:48:58 mutations that block apoptosis. All of these are important in 558 00:48:58 --> 00:49:03 cancer. Next time I'm going to tell you that 559 00:49:03 --> 00:49:07 some cancers are caused by inherited mutations. And this is a pedigree 560 00:49:07 --> 00:49:12 of such an example, and lastly to remind you, 561 00:49:12 --> 00:49:16 mutations affect proliferation in cell death. But they also affect 562 00:49:16 --> 00:49:21 many other things: invasion, angiogenesis, as well as metastasis. 563 00:49:21 --> 49:24 And I'll stop there.