1 00:00:00,400 --> 00:00:04,240 *Ambient music plays and fades out* 2 00:00:05,000 --> 00:00:06,920 Hi. Thank you for listening. 3 00:00:07,040 --> 00:00:09,320 My name is Britt Duijndam. In this presentation... 4 00:00:09,440 --> 00:00:11,840 I would like to give you an overview of my PhD project... 5 00:00:11,960 --> 00:00:16,440 where we developed a reporter platform for oestrogen receptor alpha signaling. 6 00:00:17,600 --> 00:00:20,280 Exposure to certain pharmaceuticals or compounds... 7 00:00:20,400 --> 00:00:22,960 can result in tumour development in humans. 8 00:00:23,080 --> 00:00:27,600 Whereas genotoxic compounds can be readily identified in vitro... 9 00:00:27,720 --> 00:00:35,480 we mostly rely on lifetime rodent bioassays for the detection of non-genotoxic carcinogens. 10 00:00:35,600 --> 00:00:40,640 However, this lifetime rodent bioassay has its limitations. 11 00:00:40,760 --> 00:00:42,720 In line with the 3R-principle... 12 00:00:42,840 --> 00:00:46,960 known as the replacement, reduction and refinement of animal testing... 13 00:00:47,080 --> 00:00:51,000 more efforts are being made to develop cell-based assays. 14 00:00:51,120 --> 00:00:53,920 But since the mode of action of these non-genotoxic carcinogens... 15 00:00:54,040 --> 00:00:58,400 is quite complex and diverse, this remains challenging. 16 00:00:59,520 --> 00:01:05,240 Whereas genotoxic compounds mostly rely on DNA damage as their mode of action... 17 00:01:05,360 --> 00:01:08,600 the mode of action of non-genotoxic carcinogens is diverse. 18 00:01:08,720 --> 00:01:14,120 And we wanted to focus on receptor-mediated endocrine modification. 19 00:01:14,240 --> 00:01:17,200 This receptor binding can have various consequences... 20 00:01:17,320 --> 00:01:23,880 such as the induction of cellular processes such as proliferation, apoptosis, homeostasis... 21 00:01:24,000 --> 00:01:27,080 and this can eventually culminate in tumour development. 22 00:01:28,560 --> 00:01:32,200 In this project, we studied one of these involved receptors... 23 00:01:32,320 --> 00:01:34,720 namely the oestrogen receptor alpha. 24 00:01:36,960 --> 00:01:43,200 To evaluate and link this complex ERa signaling pathway to non-genotoxic carcinogenicity... 25 00:01:43,320 --> 00:01:46,640 we made use of an adverse outcome pathway framework. 26 00:01:46,760 --> 00:01:51,720 This AOP is a simplified presentation of a biological process... 27 00:01:51,840 --> 00:01:55,920 which describes the link between the initial chemical interaction... 28 00:01:56,040 --> 00:02:00,000 and the occurrence of adverse effects, by using a series of key events. 29 00:02:01,800 --> 00:02:09,040 When we fill out this AOP for ERa activation in relation to proliferation, it will look like this. 30 00:02:09,160 --> 00:02:14,040 So as a molecular initiating event, the receptor will be bound by a compound... 31 00:02:14,160 --> 00:02:17,920 translocate to the nucleus, where it acts as a transcription factor. 32 00:02:18,040 --> 00:02:23,720 This will lead to the first key event, namely the transcription of target genes... 33 00:02:23,840 --> 00:02:28,760 followed by subsequent key events like cell cycle progression and proliferation... 34 00:02:28,880 --> 00:02:32,880 ultimately culminating in tumour formation. 35 00:02:33,000 --> 00:02:36,080 To integrate this AOP into human risk assessment... 36 00:02:36,200 --> 00:02:41,520 it is essential to carefully define and quantify these key events. 37 00:02:41,640 --> 00:02:45,840 For this purpose, we established fluorescent reporters... 38 00:02:45,960 --> 00:02:48,560 which in combination with advanced live cell imaging... 39 00:02:48,680 --> 00:02:53,240 allow for time- and concentration-dependent assessment of pathway activation. 40 00:02:55,400 --> 00:02:58,960 To monitor the first key event, target activation... 41 00:02:59,080 --> 00:03:06,360 we carefully identify three target genes, namely GREB1, PGR and TFF1. 42 00:03:06,480 --> 00:03:13,000 These targets fit in our AOP because they are inducible by our model compound E2... 43 00:03:13,120 --> 00:03:16,720 which is also known as 17ß-estradiol. 44 00:03:16,840 --> 00:03:19,920 This is the endogenous ligand of the oestrogen receptor alpha... 45 00:03:20,040 --> 00:03:24,000 and also a known human non-genotoxic carcinogen. 46 00:03:24,120 --> 00:03:28,720 Secondly, this induction is completely dependent on oestrogen receptor alpha... 47 00:03:28,840 --> 00:03:34,040 and we have demonstrated that they play a role in E2-induced proliferation. 48 00:03:35,760 --> 00:03:42,280 With BAC-GFP technology, we've coupled these three target genes to a green fluorescent protein. 49 00:03:42,400 --> 00:03:45,320 These constructs make use of an endogenous promoter. 50 00:03:45,440 --> 00:03:49,960 This better reflects the regulation of these targets in the human situation. 51 00:03:50,080 --> 00:03:54,480 Next, we transfected these constructs in human MCF7 cells. 52 00:03:54,600 --> 00:03:58,720 In this way, we developed three different fluorescent reporters. 53 00:03:58,840 --> 00:04:01,440 In combination with advanced live cell imaging... 54 00:04:01,560 --> 00:04:05,600 we can monitor the dynamic of pathway activation in these individual reporters... 55 00:04:05,720 --> 00:04:07,600 on a single-cell level. 56 00:04:07,720 --> 00:04:12,280 When we quantify the GFP expression over time, displayed in the graphs... 57 00:04:12,400 --> 00:04:16,400 the three reporters demonstrate concentration-dependent activation. 58 00:04:16,520 --> 00:04:20,720 However, they show clearly distinct activation dynamic patterns over time. 59 00:04:21,840 --> 00:04:26,840 Now that we have covered the first key event, we move on to the second key event. 60 00:04:26,960 --> 00:04:32,440 To monitor cell cycle progression, we developed another reporter in the MCF7 cells... 61 00:04:32,560 --> 00:04:35,080 which is based on a cell cycle indicator construct. 62 00:04:35,200 --> 00:04:40,200 We named this cell line, or this reporter, we named it MCF7-FUCCI. 63 00:04:40,320 --> 00:04:46,240 This FUCCI reporter makes use of two different genes coupled to two different fluorescent tags. 64 00:04:46,360 --> 00:04:52,040 Geminin coupled to GFP and Cdt1 coupled to RFP. 65 00:04:52,160 --> 00:04:56,160 These genes have distinct expression patterns in the cell cycle. 66 00:04:56,280 --> 00:04:58,600 So based on the expression of the tags... 67 00:04:58,720 --> 00:05:02,440 we can determine in which phase a cell is residing over time. 68 00:05:02,560 --> 00:05:07,760 We employed this FUCCI reporter to look at the link between target activation... 69 00:05:07,880 --> 00:05:09,680 and cell cycle progression. 70 00:05:09,800 --> 00:05:12,440 By using small interfering RNAs... 71 00:05:12,560 --> 00:05:16,880 we knock down the expression of the individual target genes in the FUCCI reporter... 72 00:05:17,000 --> 00:05:21,920 expose the FUCCI reporter to E2 and monitor the cell cycle progression over time. 73 00:05:22,920 --> 00:05:29,040 As expected, cell cycle progression is inhibited after knockdown of these target genes... 74 00:05:29,160 --> 00:05:31,480 which can be seen in the time-lapse movies. 75 00:05:31,600 --> 00:05:36,400 Whereas control cells keep progressing through the cell cycle upon exposure to E2... 76 00:05:36,520 --> 00:05:43,400 cells with knockdown of PGR or TFF1 tend to reside for a longer period in the G1 phase. 77 00:05:43,520 --> 00:05:51,040 Interestingly, cells with knockdown of GREB1 tend to accumulate in the G1-S transition phase. 78 00:05:51,160 --> 00:05:53,120 This is also reflected in the graph... 79 00:05:53,240 --> 00:06:00,040 where we show the fraction of cells in each cell cycle phase at the end of our experiment. 80 00:06:00,160 --> 00:06:03,120 This clearly indicates that these genes have a different role... 81 00:06:03,240 --> 00:06:05,560 in cell cycle progression and proliferation. 82 00:06:05,680 --> 00:06:09,640 Currently, we're further looking into this role of the genes... 83 00:06:09,760 --> 00:06:15,160 by using whole transcriptome next generation sequencing. 84 00:06:15,280 --> 00:06:19,960 As I hopefully demonstrated, we've established a human cell-based reporter platform... 85 00:06:20,080 --> 00:06:25,280 which can provide us more insight in pro-proliferative ERa signaling... 86 00:06:25,400 --> 00:06:29,080 by covering different key events in the pathway. 87 00:06:29,200 --> 00:06:33,560 But can this reporter platform also detect other estrogenic compounds... 88 00:06:33,680 --> 00:06:35,960 besides our model compound E2? 89 00:06:36,080 --> 00:06:40,600 And is it able to discriminate between non-estrogenic and estrogenic compounds? 90 00:06:42,320 --> 00:06:48,240 For this purpose, we used a reference set as defined by the OECD test guideline. 91 00:06:48,360 --> 00:06:50,520 This list of reference compounds... 92 00:06:50,640 --> 00:06:55,120 is composed of a wide variety of non-estrogenic and estrogenic compounds... 93 00:06:55,240 --> 00:07:02,080 ranging from very potent pharmaceuticals to very weak industrial phenolics. 94 00:07:02,200 --> 00:07:04,720 We've exposed our four reporters... 95 00:07:04,840 --> 00:07:10,160 and collected concentration response curves at different time points for each reporter. 96 00:07:11,200 --> 00:07:15,800 In this way, we monitor the different key events of the pathway. 97 00:07:15,920 --> 00:07:20,920 Namely, target activation by monitoring GFP intensity... 98 00:07:21,040 --> 00:07:24,160 FUCCI fractions reflecting cell cycle progression... 99 00:07:24,280 --> 00:07:30,440 and additionally, we determined the total amount of cells to reflect proliferation. 100 00:07:30,560 --> 00:07:34,600 Based on the generated concentration response curves... 101 00:07:34,720 --> 00:07:39,880 we determined the potency of each compound reflected by EC50 value. 102 00:07:40,000 --> 00:07:42,840 When we incorporate these EC50 values... 103 00:07:42,960 --> 00:07:49,440 of each compound for each reporter at a different time point in one single heat map... 104 00:07:49,560 --> 00:07:54,400 we are clearly able to discriminate non-estrogenic from estrogenic compounds... 105 00:07:54,520 --> 00:08:01,880 and we can cluster the estrogenic compounds based on their potency in different clusters. 106 00:08:02,000 --> 00:08:06,240 Finally, we also compared the performance of our reporters... 107 00:08:06,360 --> 00:08:10,000 to other OECD-validated assays. 108 00:08:10,120 --> 00:08:15,640 Based on the EC50 values, our reporters are equally sensitive as other assays. 109 00:08:15,760 --> 00:08:18,800 To summarize, our human cell-based reporter platform... 110 00:08:18,920 --> 00:08:22,880 can monitor ERa pathway activation on a single cell level... 111 00:08:23,000 --> 00:08:26,840 and by additionally monitoring cell cycle progression and proliferation... 112 00:08:26,960 --> 00:08:31,120 we can link activation of the pathway to a potential adverse outcome. 113 00:08:31,240 --> 00:08:36,520 By making use of endogenous regulation, this better reflects the human situation. 114 00:08:36,640 --> 00:08:41,840 Besides that, it can identify various estrogenic compounds with different potencies... 115 00:08:41,960 --> 00:08:46,640 with a similar sensitivity as other OECD-validated assays. 116 00:08:47,760 --> 00:08:50,600 All in all, it represents a weight-of-evidence approach... 117 00:08:50,720 --> 00:08:56,360 which provides a better understanding of ERa pathway activation in the context of proliferation. 118 00:08:56,480 --> 00:09:00,640 In turn, this will yield in improved risk assessment for human health. 119 00:09:00,960 --> 00:09:03,960 *Ambient music plays and fades out*