1 00:00:15 --> 00:00:20 And he took what was left, which much be very, very small, 2 00:00:20 --> 00:00:25 smaller than the size of a cell because the cells were removed by 3 00:00:25 --> 00:00:30 filtration, and he injected it into another bird. Now, a healthy bird. 4 00:00:30 --> 00:00:35 And strikingly that bird developed a tumor. So he was able to transfer 5 00:00:35 --> 00:00:40 whatever agent it was that was associated with tumor development 6 00:00:40 --> 00:00:46 from this bird to this bird. He concluded that it was something 7 00:00:46 --> 00:00:51 smaller than a cell, and he concluded that it was a virus. 8 00:00:51 --> 00:00:57 And indeed he was right. Now, the tumor that these birds 9 00:00:57 --> 00:01:02 developed is called a sarcoma. It's a tumor of the muscle in this 10 00:01:02 --> 00:01:07 case. And the virus that was responsible for this tumor was named 11 00:01:07 --> 00:01:12 after Rous, and it goes by the name of Rous sarcoma virus or RSV. 12 00:01:12 --> 00:01:17 Now, there was great skepticism about the connections between 13 00:01:17 --> 00:01:22 viruses and cancer at that time, and actually for many decades 14 00:01:22 --> 00:01:27 thereafter. And so Rous' work was not fully appreciated 15 00:01:27 --> 00:01:32 for some time. But it was eventually appreciated. 16 00:01:32 --> 00:01:37 And Rous actually won the Nobel Prize 50 years after he made this 17 00:01:37 --> 00:01:41 discovery, when it was confirmed that indeed the stuff that he was 18 00:01:41 --> 00:01:46 studying was highly relevant to the situation in humans. 19 00:01:46 --> 00:01:51 Not analogous, necessarily, but highly relevant. Over the years, 20 00:01:51 --> 00:01:55 since that discovery and discoveries made by others, 21 00:01:55 --> 00:02:00 demonstrated that the Rous sarcoma virus viral genome carried a set of 22 00:02:00 --> 00:02:09 genes that were familiar. 23 00:02:09 --> 00:02:12 These are the same sorts of genes carried by many viruses of this 24 00:02:12 --> 00:02:16 class. Rous sarcoma virus happens to be a retrovirus. 25 00:02:16 --> 00:02:20 I'll teach you more about those in a future lecture. 26 00:02:20 --> 00:02:24 It's the same general class that includes HIV. And there were some 27 00:02:24 --> 00:02:28 familiar genes that were responsible for the replication functions 28 00:02:28 --> 00:02:32 of the virus. But then there was a new gene 29 00:02:32 --> 00:02:36 present in this strain of the virus, specific to Rous sarcoma virus, 30 00:02:36 --> 00:02:41 which was named Src and was later considered to be the relevant 31 00:02:41 --> 00:02:45 oncogene, the gene that was responsible for the tumor forming 32 00:02:45 --> 00:02:50 capabilities of this Rous sarcoma virus. So there was then great 33 00:02:50 --> 00:02:54 interest in what was this Src gene? What was this special gene that 34 00:02:54 --> 00:02:59 could cause normal cells to become cancer cells? 35 00:02:59 --> 00:03:02 And research then went on for, again, a number of years studying 36 00:03:02 --> 00:03:06 Src, studying its biochemical functions, but the real question 37 00:03:06 --> 00:03:10 that was of great interest over the years was where did Src come from? 38 00:03:10 --> 00:03:20 Most viruses in this class don't 39 00:03:20 --> 00:03:24 carry Src, and it wasn't at all clear what the origins of this 40 00:03:24 --> 00:03:28 cancer-causing gene were. Was it a rare viral gene or perhaps 41 00:03:28 --> 00:03:32 did it come from the host cells themselves? 42 00:03:32 --> 00:03:37 And in experiments that were done, actually by my PhD advisor, Harold 43 00:03:37 --> 00:03:42 Varmus and his colleague Mike Bishop, it was shown that the Src gene 44 00:03:42 --> 00:03:47 actually does indeed reside in the cells of the host and gets picked up 45 00:03:47 --> 00:03:53 by a recombination process by the virus. And I'll just illustrate 46 00:03:53 --> 00:03:58 that for you. It turns out that there's a related virus fairly 47 00:03:58 --> 00:04:04 common in chickens called avian leukosis virus. 48 00:04:04 --> 00:04:09 And this is the predecessor of Rous sarcoma virus. 49 00:04:09 --> 00:04:14 It's a virus that has in its genome just the replication genes. 50 00:04:14 --> 00:04:19 And I'll just draw a viral capsid around this virus. 51 00:04:19 --> 00:04:24 So this is the avian leukosis virus with its genome and replication 52 00:04:24 --> 00:04:29 genes. Now, what they showed was that the avian leukosis virus, 53 00:04:29 --> 00:04:34 when it infects cells, here's a normal chicken cell. 54 00:04:34 --> 00:04:42 Most of the time it just makes more 55 00:04:42 --> 00:04:47 copies of itself like viruses will do. But occasionally, 56 00:04:47 --> 00:04:53 through a recombination mechanism, the avian leukosis virus will 57 00:04:53 --> 00:04:58 actually pick up a gene which is present in the normal genome of the 58 00:04:58 --> 00:05:03 chicken, a gene which looks very much like the Src gene present in 59 00:05:03 --> 00:05:08 Rous sarcoma virus. And through this recombination 60 00:05:08 --> 00:05:14 mechanism, the details of which I won't go through, 61 00:05:14 --> 00:05:19 very rarely you'll get a recombinant virus produced which has in its 62 00:05:19 --> 00:05:25 genome the replication genes, but now also Src gene. And this 63 00:05:25 --> 00:05:30 virus, which modifies the Src gene slightly in the course of the 64 00:05:30 --> 00:05:36 development of the virus, now, when introduced into birds, 65 00:05:36 --> 00:05:41 will very efficiently cause tumors. And what this showed, 66 00:05:41 --> 00:05:45 what this actually Nobel Prize winning experiment showed was that 67 00:05:45 --> 00:05:49 the oncogene, powerful cancer-causing gene resides in the 68 00:05:49 --> 00:05:53 DNA of the chicken. And actually there were similar 69 00:05:53 --> 00:05:57 genes, very analogous genes residing in the DNA of all vertebrate species, 70 00:05:57 --> 00:06:03 including humans. You are sitting there with two 71 00:06:03 --> 00:06:09 copies of the Src gene inside of you. So a question is, 72 00:06:09 --> 00:06:16 if that's true, if we all have these oncogenes in our cells why don't we 73 00:06:16 --> 00:06:23 get cancer? Why aren't we all sitting there as one large sarcoma? 74 00:06:23 --> 00:06:30 Anybody? Well, there are two answers. Yeah? Somebody. 75 00:06:30 --> 00:06:34 Claudette is pointing to somebody. Am I blind? Oh, hi. Excellent. 76 00:06:34 --> 00:06:38 Yes. So what's different about the situation in the chicken cell and 77 00:06:38 --> 00:06:42 the situation in the viral genome is how the gene is expressed. 78 00:06:42 --> 00:06:46 Here it might be expressed properly under what we would call 79 00:06:46 --> 00:06:50 physiological control, express where it's supposed to be 80 00:06:50 --> 00:06:54 when it's supposed to be. And here it's been removed from 81 00:06:54 --> 00:06:58 that regulatory network and is expressed perhaps at two high levels 82 00:06:58 --> 00:07:03 at the wrong time. And under those conditions it can 83 00:07:03 --> 00:07:07 push cells to inappropriately divide. So the virus has hijacked this gene 84 00:07:07 --> 00:07:11 and changes its regulation. That's one explanation. There's 85 00:07:11 --> 00:07:15 potentially another explanation for why we're not all sitting around 86 00:07:15 --> 00:07:19 with tumors, and I'll come to that in a moment. Now, 87 00:07:19 --> 00:07:23 as I said, Varmus and Bishop won the Nobel Prize for this work in 1989. 88 00:07:23 --> 00:07:27 And the day that they won the Nobel Prize Harold Varmus' sister-in-law 89 00:07:27 --> 00:07:31 was standing in a cafeteria line at Berkeley and she overheard two guys 90 00:07:31 --> 00:07:34 talking behind her. One guy said to the other, 91 00:07:34 --> 00:07:38 what are you going to have for lunch? And the other guy said, 92 00:07:38 --> 00:07:42 I don't know but it ain't going to be the chicken because two guys just 93 00:07:42 --> 00:07:46 won the Nobel Prize for showing that chicken causes cancer. 94 00:07:46 --> 00:07:50 [LAUGHTER] Which is not exactly true. But nevertheless. 95 00:07:50 --> 00:07:54 Now we know since that work that there are a large number of these 96 00:07:54 --> 00:07:58 viruses which are called collectively acutely transforming 97 00:07:58 --> 00:08:06 viruses. 98 00:08:06 --> 00:08:09 There are a large number of acutely transforming viruses that carry in 99 00:08:09 --> 00:08:13 their genomes and oncogene which they have cooperated from the host 100 00:08:13 --> 00:08:17 cell. These viruses are not viruses of human beings. 101 00:08:17 --> 00:08:21 They're viruses of experimental animals and usually generated in an 102 00:08:21 --> 00:08:25 experimental setting. Mice, rats, chickens, 103 00:08:25 --> 00:08:29 turkeys, other species have been used to generate such viruses. 104 00:08:29 --> 00:08:33 And about 50 or so oncogenes have been discovered through this context, 105 00:08:33 --> 00:08:37 including one that's hopefully familiar to you already and will 106 00:08:37 --> 00:08:42 come again, members of the Ras gene family which I've told you about as 107 00:08:42 --> 00:08:46 an important signaling molecule in mitogenic signaling pathways, 108 00:08:46 --> 00:08:51 and another important oncogene that I'll mention briefly later called 109 00:08:51 --> 00:08:55 mic, and about 50 more. And it turns out that they were a 110 00:08:55 --> 00:09:00 very useful source to identify cancer-associated genes in humans. 111 00:09:00 --> 00:09:04 Many of the genes that we now know are important in human cancer were 112 00:09:04 --> 00:09:08 initially discovered through that process. Now, 113 00:09:08 --> 00:09:12 as I said, most of the time human cancer has nothing to do with 114 00:09:12 --> 00:09:16 viruses so don't be confused. There are a few human cancers that 115 00:09:16 --> 00:09:20 are virally associated. The major one is cervical cancer. 116 00:09:20 --> 00:09:28 About 50% of cervical cancers, 117 00:09:28 --> 00:09:34 particularly in the Developing World, are associated with the virus called 118 00:09:34 --> 00:09:40 human papillomavirus or HPV, and specifically the high-risk types. 119 00:09:40 --> 00:09:46 Not all papillomaviruses will cause 120 00:09:46 --> 00:09:50 cancer. Papillomavirus is the same virus that causes warts, 121 00:09:50 --> 00:09:54 for example. And there are many papillomaviruses that are not 122 00:09:54 --> 00:09:58 associated with true malignancies, but human papillomaviruses of the 123 00:09:58 --> 00:10:01 high-risk type are. And they can give rise to cervical 124 00:10:01 --> 00:10:05 cancer. Fortunately, companies are now developing 125 00:10:05 --> 00:10:09 vaccines against HPVs which are greatly affecting the risk of 126 00:10:09 --> 00:10:13 cervical cancer around the world. So this is a major step forward in 127 00:10:13 --> 00:10:17 controlling this particular cancer type. So because most human cancers 128 00:10:17 --> 00:10:21 are not virally associated, there was still some skepticism 129 00:10:21 --> 00:10:25 about the importance of this discovery for human cancer. 130 00:10:25 --> 00:10:29 Maybe it was true of the viruses, maybe it was true of these 131 00:10:29 --> 00:10:33 experimental animals, but is it true of human beings? 132 00:10:33 --> 00:10:39 Do these oncogenes have anything to do with human cancer? 133 00:10:39 --> 00:10:45 And this debate went on for a little while longer until Bob 134 00:10:45 --> 00:10:51 Weinberg here at MIT in about 1980 did the following experiment. 135 00:10:51 --> 00:11:00 He took DNA not from a bird but from 136 00:11:00 --> 00:11:06 a human being who carried a tumor in his bladder. So this individual had 137 00:11:06 --> 00:11:12 bladder cancer. This cancer was put into cell 138 00:11:12 --> 00:11:18 culture and turned into a cancer cell line, and it was actually this 139 00:11:18 --> 00:11:24 material that Weinberg's lab worked with. And they asked the question, 140 00:11:24 --> 00:11:30 are there genes in the cancer cell line that are cancer-causing? 141 00:11:30 --> 00:11:35 Are there oncogenes that I can discover within those cancer cells? 142 00:11:35 --> 00:11:40 So the experiment that they did was to take DNA, they isolated DNA from 143 00:11:40 --> 00:11:45 the cancer cells, and they introduced it into mouse 144 00:11:45 --> 00:11:51 cells in the laboratory that were "normal". 145 00:11:51 --> 00:11:57 And what I mean by that is they 146 00:11:57 --> 00:12:01 looked pretty normal compared to the cancer cells which had a deranged 147 00:12:01 --> 00:12:05 sort of architecture, as I mentioned last time. 148 00:12:05 --> 00:12:08 They grew flat and didn't grow on top of each other like cancer cells 149 00:12:08 --> 00:12:12 will do. And, importantly, if you were to inject 150 00:12:12 --> 00:12:16 these cells into an immunocompromised mouse they 151 00:12:16 --> 00:12:19 wouldn't form a tumor. They were nontumorigenic. 152 00:12:19 --> 00:12:23 Whereas, these cells, if injected into a mouse, would form tumors. 153 00:12:23 --> 00:12:27 OK? So they isolated DNA from the cancer cells and put it 154 00:12:27 --> 00:12:31 on the mouse cells. The mouse cells will, 155 00:12:31 --> 00:12:37 at some frequency, take up the DNA, incorporate the human DNA into their 156 00:12:37 --> 00:12:43 own genomes and begin to express the genes from the human DNA. 157 00:12:43 --> 00:12:49 And they found at low frequency that within this population of 158 00:12:49 --> 00:12:55 otherwise normal-looking mouse cells abnormal colonies of "transformed" 159 00:12:55 --> 00:13:00 cells could be found. And these transformed cells looked a 160 00:13:00 --> 00:13:04 lot like the cancer cells. Their shape was different, 161 00:13:04 --> 00:13:09 they grew on top of one another, and they had other properties that 162 00:13:09 --> 00:13:13 also made them similar to cancer cells in the sense that if they 163 00:13:13 --> 00:13:17 injected these cells into an immunocompromised animal, 164 00:13:17 --> 00:13:22 whereas the normal cells would not form a tumor, these transformed 165 00:13:22 --> 00:13:26 cells would. So they were able to convert normal cells into cancer 166 00:13:26 --> 00:13:31 cells through the addition of DNA from a cancer cell line. 167 00:13:31 --> 00:13:36 So what's going on here? What did they do in this experiment? 168 00:13:36 --> 00:13:41 Yeah. They eventually did. To get to that point they were able 169 00:13:41 --> 00:13:46 to isolate from the full genome of the cancer cell an individual gene 170 00:13:46 --> 00:13:52 that had been transferred into these mouse cells. They isolated that 171 00:13:52 --> 00:13:57 human gene away from the mouse genes, and then they preformed 172 00:13:57 --> 00:14:02 DNA sequencing. And they discovered the first human 173 00:14:02 --> 00:14:06 oncogene that was isolated from a cancer as opposed to a virus. 174 00:14:06 --> 00:14:10 And, as I alluded to last time, the gene that they isolated in this 175 00:14:10 --> 00:14:14 fashion was a familiar one. It was the Ras gene, a gene that 176 00:14:14 --> 00:14:18 had been discovered in the context of acutely transforming retroviruses. 177 00:14:18 --> 00:14:22 It was already known to be cancer-associated, 178 00:14:22 --> 00:14:26 but now it's in the context of real human cancer. And this 179 00:14:26 --> 00:14:31 was a major advance. Moreover, they sequenced the Ras 180 00:14:31 --> 00:14:35 gene from the cancer and compared it to the sequence of the Ras gene from 181 00:14:35 --> 00:14:40 normal cells and they found a difference. They found that it had 182 00:14:40 --> 00:14:44 been mutated, just as the genetic theory of cancer would predict. 183 00:14:44 --> 00:14:48 Compared to the normal sequence of the Ras gene, which is shown above, 184 00:14:48 --> 00:14:53 the sequence of the Ras gene isolated from the human bladder 185 00:14:53 --> 00:14:57 cancer sample carried a single mutation which converted a glycine 186 00:14:57 --> 00:15:02 amino acid to a valine amino acid. And the consequence of that mutation 187 00:15:02 --> 00:15:07 changed the Ras protein from being a signaling molecule that could be 188 00:15:07 --> 00:15:12 regulated from an on state to an off state to a signaling molecule that 189 00:15:12 --> 00:15:17 could not be regulated. Once it go turned on it couldn't' 190 00:15:17 --> 00:15:22 get turned off. So it was a constitutively active 191 00:15:22 --> 00:15:27 signaling molecule, which makes sense for a cancer cell 192 00:15:27 --> 00:15:32 that is continually proliferating. Now, there's an issue here that I 193 00:15:32 --> 00:15:37 would like you to ponder. And that is that this is a single 194 00:15:37 --> 00:15:42 point mutation, a single base change. 195 00:15:42 --> 00:15:47 In all of your cells, during cell division, there's a 196 00:15:47 --> 00:15:52 particular mutation rate. It's not known exactly, at least 197 00:15:52 --> 00:15:57 for all cells in your body what that rate is. But let's estimate that 198 00:15:57 --> 00:16:03 it's about ten to the minus ninth per base pair per cell division. 199 00:16:03 --> 00:16:07 Now, if you calculate how many cell divisions are going on in your body 200 00:16:07 --> 00:16:12 at any given time, you can estimate that you have about 201 00:16:12 --> 00:16:17 ten to the third to ten to the fifth Ras mutant cells inside you as you 202 00:16:17 --> 00:16:22 sit there today. Maybe as many 100, 203 00:16:22 --> 00:16:27 00. Maybe more than a million cells that carry that very mutation 204 00:16:27 --> 00:16:32 in your bodies. So I ask you again, 205 00:16:32 --> 00:16:36 why is it that you don't all have tumors? This time it's not the 206 00:16:36 --> 00:16:40 explanation that we heard before about regulation because this is the 207 00:16:40 --> 00:16:44 native gene which is being changed. It's being regulated the same way. 208 00:16:44 --> 00:16:48 So why is it that we don't all have bladder cancer, 209 00:16:48 --> 00:16:52 for example, or some other type of tumor? Well, it relates to what I 210 00:16:52 --> 00:16:56 told you last time, that cancer is rarely a single-step 211 00:16:56 --> 00:17:01 event. Cancers rarely are associated with a 212 00:17:01 --> 00:17:05 single mutation. A single mutation may be involved, 213 00:17:05 --> 00:17:09 may be involved in initiation, but it's not sufficient to give you all 214 00:17:09 --> 00:17:13 the steps you need for a full cancer. As I mentioned last time, 215 00:17:13 --> 00:17:18 you might need five, ten or twenty distinct mutations to get a true 216 00:17:18 --> 00:17:22 cancer. So you might have mutant cells in you, they might be totally 217 00:17:22 --> 00:17:26 normal, but if layered on top of those mutations are other mutations 218 00:17:26 --> 00:17:31 they could progress. OK? Now, that's particularly important 219 00:17:31 --> 00:17:35 because the mutations that we're talking about are, 220 00:17:35 --> 00:17:39 at least by some assays, dominant. And the old genetics 221 00:17:39 --> 00:17:44 terminology of dominant and recessive, these mutations are 222 00:17:44 --> 00:17:48 dominant. Note, in this assay we're transferring a 223 00:17:48 --> 00:17:53 single gene into an otherwise normal cell and seeing a phenotype. 224 00:17:53 --> 00:17:57 That's an example of dominance. And these genes can, in the right 225 00:17:57 --> 00:18:01 context, function in a dominant fashion on top of a normal copy or 226 00:18:01 --> 00:18:06 normal copies of the gene. And you'll see why a distinction 227 00:18:06 --> 00:18:10 between dominant and recessive is important in a moment. 228 00:18:10 --> 00:18:14 Now, as I hope you remember from earlier lectures about signal 229 00:18:14 --> 00:18:19 transduction, the Ras proteins fall within a signal transduction pathway 230 00:18:19 --> 00:18:23 that's very well worked out. They link transmembrane growth 231 00:18:23 --> 00:18:28 factor receptors through intermediary proteins, 232 00:18:28 --> 00:18:32 adaptors and exchange factors, to downstream components involved in, 233 00:18:32 --> 00:18:36 for example, phosphorylation kinases and transcription factors that turn 234 00:18:36 --> 00:18:41 on the expression of target genes. This is the pathway that I've 235 00:18:41 --> 00:18:45 illustrated to you before. And we now know that many of the 236 00:18:45 --> 00:18:49 components of this pathway, this so-called mitogenic signaling 237 00:18:49 --> 00:18:53 pathway that drives cells to divide are mutated in cancers. 238 00:18:53 --> 00:18:57 Not just Ras, which is actually mutated at pretty high frequency in 239 00:18:57 --> 00:19:02 cancers, but many of these components are mutated as well. 240 00:19:02 --> 00:19:06 And they're mutated through different mechanisms. 241 00:19:06 --> 00:19:10 Subtle mutations, like I've just told you about for Ras, 242 00:19:10 --> 00:19:15 where single nucleotides can be changed and a single amino acid can 243 00:19:15 --> 00:19:19 be changed to convert a normally regulated protein into an abnormally 244 00:19:19 --> 00:19:24 regulated protein like Ras. Some of the growth factor receptors 245 00:19:24 --> 00:19:28 are likewise activated by that mechanism. But there are other 246 00:19:28 --> 00:19:33 mechanisms, other activation mechanisms for cancer-associated 247 00:19:33 --> 00:19:37 genes, including gene amplification and translocation which I'll 248 00:19:37 --> 00:19:42 illustrate on the board. I hope you noticed on the right here, 249 00:19:42 --> 00:19:46 Review Sessions for Monday night for the exam on Wednesday, 250 00:19:46 --> 00:19:51 as well as Tutoring Sessions and indications of where you're supposed 251 00:19:51 --> 00:19:55 to go. So gene amplification is important in cancer. 252 00:19:55 --> 00:19:59 There are quite a few genes that are amplified in tumors compared to 253 00:19:59 --> 00:20:04 normal cells. And I'll give you one example which 254 00:20:04 --> 00:20:09 is relevant to breast cancer and ovarian cancer. 255 00:20:09 --> 00:20:14 In normal cells, in the DNA of normal cells there's a 256 00:20:14 --> 00:20:19 gene called HER2 which is one of these growth factor receptors like 257 00:20:19 --> 00:20:24 number two up there. And of course the normal cells are 258 00:20:24 --> 00:20:30 diploid so they have two copies of the HER2 gene. 259 00:20:30 --> 00:20:35 And through normal transcription and translation that's going to give 260 00:20:35 --> 00:20:41 rise to a particular concentration of the growth factor receptor on the 261 00:20:41 --> 00:20:47 surface of the cells. And that's going to give rise to a 262 00:20:47 --> 00:20:53 certain signal emanating from that growth factor receptor which will 263 00:20:53 --> 00:20:59 cause the cells to divide at the appropriate time and place. 264 00:20:59 --> 00:21:05 In about 30% of breast cancers and a similar percentage of ovarian 265 00:21:05 --> 00:21:11 cancers, the number of copies of the HER2 gene has been increased 266 00:21:11 --> 00:21:17 dramatically, sometimes as much as a hundred fold. And these are due to 267 00:21:17 --> 00:21:23 errors in DNA replication. Instead of having DNA copied once 268 00:21:23 --> 00:21:29 in this region, it gets copied again and again and 269 00:21:29 --> 00:21:34 again and again. And now, in these cancer cells, 270 00:21:34 --> 00:21:38 you don't have that same concentration of the growth factor 271 00:21:38 --> 00:21:42 receptor. You have a much higher concentration of the receptor. 272 00:21:42 --> 00:21:46 And because there's a much higher concentration of the receptor, 273 00:21:46 --> 00:21:51 you might get as much as ten or a hundred times the level of signaling. 274 00:21:51 --> 00:21:55 And the consequences of this are that a cell that shouldn't divide 275 00:21:55 --> 00:21:59 doesn't have actually enough concentration of growth factors 276 00:21:59 --> 00:22:04 where it should normally divide will now divide anyway. 277 00:22:04 --> 00:22:07 Inappropriate proliferation, the hallmark of cancer. Now, 278 00:22:07 --> 00:22:11 the good news here is that there are antibodies against this HER2 protein. 279 00:22:11 --> 00:22:14 And they are actually effective in the treatment of breast cancer. 280 00:22:14 --> 00:22:18 They can prolong the life of women with breast cancer, 281 00:22:18 --> 00:22:21 specifically those who have amplifications of this gene. 282 00:22:21 --> 00:22:25 This drug actually does nothing for women who don't have amplifications, 283 00:22:25 --> 00:22:29 but it prolongs the life of women who do. 284 00:22:29 --> 00:22:33 So that's an important link between basic biology and treatment. 285 00:22:33 --> 00:22:38 We'll talk more about those next time. Another mechanism of 286 00:22:38 --> 00:22:43 activation of oncogenes is translocation. 287 00:22:43 --> 00:22:51 I showed you last time karyotypes of 288 00:22:51 --> 00:22:55 cancer cells in which one piece of one chromosome gets linked to a 289 00:22:55 --> 00:22:59 piece of a different chromosome. The reason that happens and is 290 00:22:59 --> 00:23:03 selected for is that new regulatory networks or new genes can be 291 00:23:03 --> 00:23:08 produced by those kinds of break and joining reactions. 292 00:23:08 --> 00:23:11 And one famous one involves this gene called myc which is a 293 00:23:11 --> 00:23:15 transcription factor like the bottom of this pathway, 294 00:23:15 --> 00:23:19 like number eight there, transcription factor which is 295 00:23:19 --> 00:23:23 important in driving cells into the cell cycle. myc is normally 296 00:23:23 --> 00:23:30 expressed from a "weak promoter". 297 00:23:30 --> 00:23:33 Which means that not a whole lot of myc mRNA is produced, 298 00:23:33 --> 00:23:37 which means that not that much myc protein is produced at any given 299 00:23:37 --> 00:23:41 time in a cell. And this gives rise to regulated 300 00:23:41 --> 00:23:51 cell division. 301 00:23:51 --> 00:23:56 However, occasionally in cancers, particularly in certain types of 302 00:23:56 --> 00:24:01 leukemia and lymphoma, translocation events take place 303 00:24:01 --> 00:24:07 where a new segment of DNA is produced which joins the normal myc 304 00:24:07 --> 00:24:16 gene to a very strong promoter. 305 00:24:16 --> 00:24:20 And now, just like this gentlemen mentioned before about the RSV, 306 00:24:20 --> 00:24:25 now a gene is not regulated properly. It's now regulated, 307 00:24:25 --> 00:24:30 in this case, too strongly. So a lot of myc mRNA is produced 308 00:24:30 --> 00:24:35 which gives rise to a lot of myc protein which gives rise to 309 00:24:35 --> 00:24:44 unregulated cell growth. 310 00:24:44 --> 00:24:48 Again, the hallmark of cancer. So the DNA of tumor cells changes 311 00:24:48 --> 00:24:52 by point mutations, by gene amplifications, 312 00:24:52 --> 00:24:56 by translocations, as well as by gene deletions, 313 00:24:56 --> 00:25:00 which I haven't shown you but also occur. 314 00:25:00 --> 00:25:04 And together these mutations are found in virtually all human cancers. 315 00:25:04 --> 00:25:09 Sometimes RAS, sometimes in growth factor receptor, 316 00:25:09 --> 00:25:13 sometimes in transcription factor, but one place or another you would 317 00:25:13 --> 00:25:18 typically find a mutation in this pathway. So signaling proliferation 318 00:25:18 --> 00:25:22 is key to tumor development. And that's not too surprising given 319 00:25:22 --> 00:25:27 how we think about how cancers arise. But these oncogenes, 320 00:25:27 --> 00:25:31 these positive regulators of tumorogenesis are not the only genes 321 00:25:31 --> 00:25:36 that are important. Normal cells do, 322 00:25:36 --> 00:25:40 in fact, use these signals. And I've equated them with like the 323 00:25:40 --> 00:25:44 gas peddle on your car, go signals, which cause normal cells 324 00:25:44 --> 00:25:48 to divide when it's appropriate to divide during development or injury 325 00:25:48 --> 00:25:52 repair or cell replenishment. But there's another class of genes 326 00:25:52 --> 00:25:56 that turns on when it's appropriate for cells to stop dividing. 327 00:25:56 --> 00:26:00 So these genes are the equivalent of the stop signals which would 328 00:26:00 --> 00:26:04 impose themselves when cell division should cease. 329 00:26:04 --> 00:26:08 Now, oncogenes promote cell division. Too much oncogene product, 330 00:26:08 --> 00:26:12 a mutant oncogene product will cause normal cells to divide too often. 331 00:26:12 --> 00:26:16 But in addition to those we find mutations in the stop signals, 332 00:26:16 --> 00:26:20 the signals that would normally halt proliferation. 333 00:26:20 --> 00:26:24 And this allows an even greater accumulation of cells. 334 00:26:24 --> 00:26:28 The go signals are the equivalent of oncogene mutations and the stop 335 00:26:28 --> 00:26:32 mutations are the equivalent of another class of genes known as 336 00:26:32 --> 00:27:12 tumor suppressor genes. 337 00:27:12 --> 00:27:17 Tumor suppressor genes have been known for about 20 years now. 338 00:27:17 --> 00:27:22 And the first one comes from this tumor here. This is a child with a 339 00:27:22 --> 00:27:27 tumor called retinoblastoma. It's a tumor of the retina. It's 340 00:27:27 --> 00:27:37 actually really rare. 341 00:27:37 --> 00:27:41 It occurs in about one in 40, 00 births in the general population, 342 00:27:41 --> 00:27:46 but there are individuals who are familialy predisposed to 343 00:27:46 --> 00:27:50 retinoblastoma. And in these kids 90% of the time 344 00:27:50 --> 00:27:55 they will develop retinoblastoma actually affecting both eyes. 345 00:27:55 --> 00:28:00 So can be actually a fairly common and severe disease in that sense. 346 00:28:00 --> 00:28:05 This gene, the gene responsible for retinoblastoma was cloned, 347 00:28:05 --> 00:28:10 again in Bob Weinberg's lab several years ago. And we now know that 348 00:28:10 --> 00:28:16 it's an important cell cycle regulator, as is indicated here. 349 00:28:16 --> 00:28:21 So the retinoblastoma gene encodes a retinoblastoma protein. 350 00:28:21 --> 00:28:26 The retinoblastoma protein is called pRB. And, 351 00:28:26 --> 00:28:32 as is illustrated on this slide, the RB protein is a negative 352 00:28:32 --> 00:28:36 regulator of the cell cycle. As you recall, 353 00:28:36 --> 00:28:40 cells normally cycle from M phase to G1/S phase through again. 354 00:28:40 --> 00:28:44 And the RB protein acts normally to restrain cell division. 355 00:28:44 --> 00:28:48 It blocks cells in the G1 phase of the cell cycle when it's active. 356 00:28:48 --> 00:28:52 When cells receive signals from growth factor pathways they 357 00:28:52 --> 00:28:56 inactivate the RB gene through phosphorylation, 358 00:28:56 --> 00:29:00 the RB protein through phosphorylation. 359 00:29:00 --> 00:29:04 And this occurs by cyclin-CDK complexes, which I told you about in 360 00:29:04 --> 00:29:09 the cell cycle lectures. There are actually two different 361 00:29:09 --> 00:29:13 cyclin-CDK proteins which phosphorylate RB and inactivate it. 362 00:29:13 --> 00:29:18 So when you need to divide you inactive this brake on the cell 363 00:29:18 --> 00:29:22 cycle and the cells can divide. When you need to stop dividing you 364 00:29:22 --> 00:29:27 activate certain growth inhibitory pathways that inhibit the kinases 365 00:29:27 --> 00:29:32 that block the phosphorylation of RB so RB stays in its active state. 366 00:29:32 --> 00:29:36 And now the cells cannot divide anymore. So RB is a molecular brake 367 00:29:36 --> 00:29:41 on the cell cycle. RB mutations, and mutations in 368 00:29:41 --> 00:29:46 other components of this pathway occur in about 90% of human tumors. 369 00:29:46 --> 00:29:51 It's a very, very commonly affected pathway in tumor development. 370 00:29:51 --> 00:29:56 What kind of mutations would you expect to see in the RB gene in 371 00:29:56 --> 00:30:01 human cancer? Would you expect to see gene amplifications, 372 00:30:01 --> 00:30:06 translocations that cause too much of the RB protein to be produced? 373 00:30:06 --> 00:30:09 Does that sound right? Remember, it's a brake. 374 00:30:09 --> 00:30:12 So what kinds of mutations do you expect to find? 375 00:30:12 --> 00:30:16 Nonsense mutations. Or more generally inactivating 376 00:30:16 --> 00:30:27 mutations. 377 00:30:27 --> 00:30:34 Nonsense mutations. Maybe deletions which would remove 378 00:30:34 --> 00:30:41 the gene all together. Now, are these mutations dominant 379 00:30:41 --> 00:30:48 or recessive? Inactivating mutations are recessive. 380 00:30:48 --> 00:30:55 So what's going on here? 381 00:30:55 --> 00:30:59 We all have two copies of the RB gene, and yet we're saying that the 382 00:30:59 --> 00:31:03 mutations that take place in this gene are loss of function mutations, 383 00:31:03 --> 00:31:06 inactivating mutations. So how is it that you ever find RB 384 00:31:06 --> 00:31:15 mutations in cancer? 385 00:31:15 --> 00:31:20 Well, the answer is that one mutation is not enough. 386 00:31:20 --> 00:31:25 One mutation of one copy of the RB gene is not enough. 387 00:31:25 --> 00:31:31 For tumor suppressor genes were the mutations are recessive, 388 00:31:31 --> 00:31:37 you need two hits. Two mutational events are required 389 00:31:37 --> 00:31:43 to now fully deprive the cell of the brake. One mutation is not enough. 390 00:31:43 --> 00:31:50 And this is now known in the field as the two-hit model of tumor 391 00:31:50 --> 00:31:56 development, at least for tumor suppressor genes. And it's 392 00:31:56 --> 00:32:02 illustrated here. Because we carry two copies of all 393 00:32:02 --> 00:32:06 of our genes, two mutational events are required to eliminate the 394 00:32:06 --> 00:32:10 function of a tumor suppressor gene. And this is what it looks like. If 395 00:32:10 --> 00:32:14 this is a normal cell which has two normal copies of the RB gene, 396 00:32:14 --> 00:32:18 the first thing to happen is that one copy of the RB gene incurs a 397 00:32:18 --> 00:32:22 mutation. It might be a nonsense mutation or a deletion, 398 00:32:22 --> 00:32:26 but whatever it is it eliminates the function of the RB gene. 399 00:32:26 --> 00:32:30 But this cell itself is normal because it still has one normal copy 400 00:32:30 --> 00:32:34 of the RB gene and this is a recessive mutation. 401 00:32:34 --> 00:32:38 So this cell here is actually normal, but it's one step away from lacking 402 00:32:38 --> 00:32:43 the RB gene all together. And there are a number of ways that 403 00:32:43 --> 00:32:47 that second copy of the gene can be lost. And they're illustrated by 404 00:32:47 --> 00:32:52 these different arrows. The simplest thing to think about 405 00:32:52 --> 00:32:56 is that that second copy of the RB gene picks up its own mutation. 406 00:32:56 --> 00:33:00 We call that a de novo mutation. And now you have a cell which has 407 00:33:00 --> 00:33:04 two independently mutated copies of RB, and that's a cell that now has 408 00:33:04 --> 00:33:08 lost control of proliferation. But there are other mechanisms, 409 00:33:08 --> 00:33:12 which are illustrated here, where the second copy of the RB gene is 410 00:33:12 --> 00:33:16 actually lost all together because the chromosome that carries that 411 00:33:16 --> 00:33:20 gene is itself lost giving rise to a cell that only has one copy of the 412 00:33:20 --> 00:33:24 chromosome. And it's the one that has the mutant RB gene. 413 00:33:24 --> 00:33:28 Or there's a recombination event which replaces the good copy of the 414 00:33:28 --> 00:33:33 RB gene with the bad copy of the RB gene giving rise to a cell that is 415 00:33:33 --> 00:33:38 lacking functional RB once again. And this term here is called loss 416 00:33:38 --> 00:33:54 of heterozygosity or LOH. 417 00:33:54 --> 00:33:57 And it's a hallmark of tumor suppressor gene mutations. 418 00:33:57 --> 00:34:00 It's an indication that there's a tumor suppressor gene mutation 419 00:34:00 --> 00:34:05 in the region. It's usually detected not by a 420 00:34:05 --> 00:34:11 change in the tumor suppressor gene itself but rather by some linked 421 00:34:11 --> 00:34:17 polymorphic marker like a SNP which is close by to the RB gene or 422 00:34:17 --> 00:34:23 whatever tumor suppressor gene it is. This individual has a big A, 423 00:34:23 --> 00:34:30 little A, and therefore is heterozygous. 424 00:34:30 --> 00:34:34 He or she is heterozygous, big A, little A. But if you notice 425 00:34:34 --> 00:34:38 in the tumors, if the tumors arise by chromosome 426 00:34:38 --> 00:34:42 loss or by mitotic recombination, the individual has lost the little A 427 00:34:42 --> 00:34:47 allele. Either there's only big A or there are two copies of big A, 428 00:34:47 --> 00:34:51 in which case we've seen loss of heterozygosity, 429 00:34:51 --> 00:34:55 LOH. And that's a common feature of tumor suppressor gene mutations. 430 00:34:55 --> 00:35:00 And I suspect you'll hear about it in problem sets and beyond. 431 00:35:00 --> 00:35:04 So LOH is an important mechanism which allows us to go from one hit 432 00:35:04 --> 00:35:08 to two hits. OK. Now, RB is not the only tumor 433 00:35:08 --> 00:35:13 suppressor gene that's mutated in cancer. And proliferation is not 434 00:35:13 --> 00:35:17 the only process that affected in tumorigenesis. 435 00:35:17 --> 00:35:22 As I told you, at the end of the day in cancer we 436 00:35:22 --> 00:35:26 care about how many cells we have. And you can have too many cells by 437 00:35:26 --> 00:35:31 increasing proliferation. And the RAS mutations and RB loss 438 00:35:31 --> 00:35:35 are examples of how you can have too much proliferation. 439 00:35:35 --> 00:35:39 But you can also have too little cell death. And we actually have 440 00:35:39 --> 00:35:43 examples of both oncogenes, which are activated to prevent cell 441 00:35:43 --> 00:35:47 death, and tumor suppressor genes that are lost to prevent cell death. 442 00:35:47 --> 00:35:52 So let me tell you a little bit about those. 443 00:35:52 --> 00:36:02 The first gene that was associated 444 00:36:02 --> 00:36:06 with apoptosis and cancer was this gene called Bcl2. 445 00:36:06 --> 00:36:11 It's related to one of the first apoptosis genes discovered in the 446 00:36:11 --> 00:36:16 Horvitz lab here at MIT, and it's involved in regulating 447 00:36:16 --> 00:36:20 caspases. You've probably forgotten by now but caspases are proteases 448 00:36:20 --> 00:36:25 that are kept normally inside your cells in an inactive state. 449 00:36:25 --> 00:36:30 And they get activated by cell death signals. 450 00:36:30 --> 00:36:36 Signals that would trigger the cells to commit apoptosis which causes the 451 00:36:36 --> 00:36:44 caspases to become active. 452 00:36:44 --> 00:36:50 And this then leads to the program of cell death. 453 00:36:50 --> 00:36:56 The Bcl2 gene functions to block, downstream of the death signals, the 454 00:36:56 --> 00:37:02 activation of caspases. Is Bcl2 an oncogene or a tumor 455 00:37:02 --> 00:37:07 suppressor gene? Does it get activated during cancer 456 00:37:07 --> 00:37:13 or inactivated during cancer? Activated. You want to suppress 457 00:37:13 --> 00:37:19 cell death in tumorigenesis. And Bcl2 does that, so Bcl2 gets 458 00:37:19 --> 00:37:25 activated. It's an oncogene. And it's typically activated by 459 00:37:25 --> 00:37:31 translocation, in the cases where it is involved. 460 00:37:31 --> 00:37:36 Translocation, like with myc, too much Bcl2, 461 00:37:36 --> 00:37:41 inappropriate cell survival. Another very important gene in 462 00:37:41 --> 00:37:47 tumorigenesis that regulates apoptosis is p53. 463 00:37:47 --> 00:38:01 p53 is actually the most commonly 464 00:38:01 --> 00:38:07 affected gene in all human cancer. 50% of all tumors -- 465 00:38:07 --> 00:38:17 -- carry p53 mutations. 466 00:38:17 --> 00:38:22 That's a remarkable number. And p53, we now know, is an 467 00:38:22 --> 00:38:27 important regulator of apoptosis. It doesn't *only* regulate 468 00:38:27 --> 00:38:33 apoptosis but it is an important regulator of apoptosis. 469 00:38:33 --> 00:38:37 p53 is like a molecular policeman inside your cells. 470 00:38:37 --> 00:38:41 It's present at very low levels normally, and it's responsive to 471 00:38:41 --> 00:38:46 various stress signals. When cells get stressed, 472 00:38:46 --> 00:38:50 for example when their DNA gets damaged or they recognize that 473 00:38:50 --> 00:38:55 they're proliferating abnormally or they're starved of oxygen, 474 00:38:55 --> 00:38:59 the levels of p53 protein go way up. And when that happens the cells 475 00:38:59 --> 00:39:04 either arrest to try to repair the damage or they will commit suicide 476 00:39:04 --> 00:39:08 killing themselves so they cannot go on to become life-threatening 477 00:39:08 --> 00:39:13 tumors. So is p53 an oncogene or a tumor 478 00:39:13 --> 00:39:17 suppressor gene? Is it activated in cancer or 479 00:39:17 --> 00:39:21 inactivated in cancer? Inactivated. It's a tumor 480 00:39:21 --> 00:39:25 suppressor gene. Cancer cells don't want to die so 481 00:39:25 --> 00:39:30 they get rid of p53 to prevent the death. 482 00:39:30 --> 00:39:34 So p53 is a tumor suppressor gene and a very frequently mutated one in 483 00:39:34 --> 00:39:39 human cancers. You can study p53 in a variety of 484 00:39:39 --> 00:39:44 ways. My lab has been studying it for a dozen years using mice that 485 00:39:44 --> 00:39:48 carry mutations in p53 that were generated by gene targeting 486 00:39:48 --> 00:39:53 technology. You don't need to know the details of this, 487 00:39:53 --> 00:39:58 but suffice to say it's possible to create mice which are either 488 00:39:58 --> 00:40:03 heterozygous for a p53 mutation or homozygous for a p53 mutation. 489 00:40:03 --> 00:40:07 And the question we asked in those experiments is would these 490 00:40:07 --> 00:40:12 heterozygous mutant mice or homozygous mutant mice be cancer 491 00:40:12 --> 00:40:17 prone? Should they be? Would you expect an animal that's 492 00:40:17 --> 00:40:21 lacking p53, one copy or both, to be cancer prone? It's a tumor 493 00:40:21 --> 00:40:26 suppressor gene so, yeah, absolutely. And sure enough 494 00:40:26 --> 00:40:32 it is. If you look at a population of mice 495 00:40:32 --> 00:40:39 and plot their percent survival as a function of time, 496 00:40:39 --> 00:40:45 one year, two years, three years, mice live about three 497 00:40:45 --> 00:40:52 years, normal mice with two copies of p53 will live a normal lifespan. 498 00:40:52 --> 00:40:59 Mice that have one functional copy of p53 die sooner. 499 00:40:59 --> 00:41:05 They live about a year and a half. And if you look in the tumors of 500 00:41:05 --> 00:41:10 these mice, the normal copy of p53 is missing, which is the prediction 501 00:41:10 --> 00:41:15 of the two-hit model of tumorigenesis. 502 00:41:15 --> 00:41:20 You need to get rid of both copies. And p53 minus-minus mice only make 503 00:41:20 --> 00:41:25 it about four to six months and die from cancer. So p53 is an 504 00:41:25 --> 00:41:30 incredibly important tumor suppressor gene. 505 00:41:30 --> 00:41:34 If you're mutated in it, your risk of getting cancer goes way, 506 00:41:34 --> 00:41:39 way up. OK. Now, in the last five minutes I just want 507 00:41:39 --> 00:41:44 to mention the fact that cancer, while typically a sporadic disease -- 508 00:41:44 --> 00:42:17 -- by which I mean there's 509 00:42:17 --> 00:42:27 no family history -- 510 00:42:27 --> 00:42:34 -- about 5% of the time there's familial predisposition. 511 00:42:34 --> 00:42:42 Cancer gets passed through the 512 00:42:42 --> 00:42:46 family, through the generations. And you can see that in this 513 00:42:46 --> 00:42:49 pedigree here. This is actually a pedigree of 514 00:42:49 --> 00:42:53 familial retinoblastoma. The individuals who inherit the 515 00:42:53 --> 00:42:56 mutant copy of the gene most of the time will develop the tumor, 516 00:42:56 --> 00:43:00 and they'll pass along that predisposition to their children. 517 00:43:00 --> 00:43:05 If you were to look at this pedigree for a while, you would see that it 518 00:43:05 --> 00:43:10 looks like an autosomal dominant pedigree, if you remember back to 519 00:43:10 --> 00:43:15 that portion of the course. If you inherit the mutation, 520 00:43:15 --> 00:43:20 you have a high likelihood of developing the tumor. 521 00:43:20 --> 00:43:25 And it doesn't matter whether the mutation is coming from your mother 522 00:43:25 --> 00:43:30 or father or whether you're a boy or a girl. What's surprising, 523 00:43:30 --> 00:43:35 despite that fact, is that most familial cancer syndromes like that 524 00:43:35 --> 00:43:40 one are caused by tumor suppressor gene mutations. 525 00:43:40 --> 00:43:51 So what's being passed through the 526 00:43:51 --> 00:43:55 generations here is a mutant copy of a tumor suppressor gene, 527 00:43:55 --> 00:43:59 the RB gene, or the breast cancer susceptibility gene, 528 00:43:59 --> 00:44:03 or colon cancer susceptibility gene. A loss of function copy of that gene 529 00:44:03 --> 00:44:08 such that these individuals who are developing cancer and passing the 530 00:44:08 --> 00:44:13 mutation onto their kids are heterozygotes. 531 00:44:13 --> 00:44:18 And, as you think about it, you have to wonder why are they 532 00:44:18 --> 00:44:23 cancer prone? Most of their cells should be normal because most of 533 00:44:23 --> 00:44:28 their cells are heterozygous for the mutation. 534 00:44:28 --> 00:44:32 But, importantly, those cells are one step away from 535 00:44:32 --> 00:44:36 mutating the other copy of the gene. In contrast to the general 536 00:44:36 --> 00:44:40 population, it carries two normal copies of the tumor suppressor gene. 537 00:44:40 --> 00:44:45 And in whose cells two mutational events have to happen in succession 538 00:44:45 --> 00:44:49 to mutate the first copy and then the second copy. 539 00:44:49 --> 00:44:53 In these people all of their cells carry the first mutation. 540 00:44:53 --> 00:44:58 They're only one mutational event away. 541 00:44:58 --> 00:45:03 And the likelihood that that will happen in the retinas of their eyes 542 00:45:03 --> 00:45:08 or in the developing breast or colon is sufficiently high that there is a 543 00:45:08 --> 00:45:13 near certainty that they will develop cancer. 544 00:45:13 --> 00:45:18 OK? So what event must happen during tumor development? 545 00:45:18 --> 00:45:23 A mutation in the second copy of the gene, one of these LOH events or 546 00:45:23 --> 00:45:28 a second de novo mutation. I also wonder what might explain 547 00:45:28 --> 00:45:33 the fact that this gentleman here, who must have the mutation because 548 00:45:33 --> 00:45:38 he passed it onto his sons, did not himself develop the tumor. 549 00:45:38 --> 00:45:42 Why is that? What's going on in that guy? Excellent. 550 00:45:42 --> 00:45:47 That's exactly the way I phrase it, he was lucky. In his developing 551 00:45:47 --> 00:45:51 retina by chance no cell underwent the second hit, 552 00:45:51 --> 00:45:56 and so his cells were just like normal and he developed normally, 553 00:45:56 --> 00:46:00 but he still passed the mutation onto his children and they 554 00:46:00 --> 00:46:05 weren't so lucky. And, finally, what would happen if 555 00:46:05 --> 00:46:09 two individuals who were heterozygous produced a homozygous 556 00:46:09 --> 00:46:13 mutant offspring? What do you think would happen in 557 00:46:13 --> 00:46:18 those individuals? Well, if they survive they should 558 00:46:18 --> 00:46:22 be mighty cancer prone because no mutational event is required to 559 00:46:22 --> 00:46:26 eliminate the function in them. They might look like these p53 560 00:46:26 --> 00:46:31 minus-minus mice. In fact, most tumor suppressor genes 561 00:46:31 --> 00:46:35 are required for normal development. So you cannot do that experiment. 562 00:46:35 --> 00:46:40 The embryo would never survive because the gene is important for 563 00:46:40 --> 00:46:44 proliferation control all over the place. So the embryo doesn't make 564 00:46:44 --> 00:46:49 it. So most of the time you cannot do that experiment. 565 00:46:49 --> 00:46:53 And, finally, I'd mentioned last time and I just want to emphasize 566 00:46:53 --> 00:46:58 that the genes that we've focused on for you in this class are genes that 567 00:46:58 --> 00:47:03 involve proliferation control and cell death. 568 00:47:03 --> 00:47:07 And we'd mentioned briefly DNA damage control. 569 00:47:07 --> 00:47:12 But there are many other processes that are important in tumorigenesis 570 00:47:12 --> 00:47:16 that are responsive to mutations in other kinds of genes like the 571 00:47:16 --> 00:47:21 process of invasion and angiogenesis and the all important process of 572 00:47:21 --> 00:47:24 metastasis. So I'll stop there.