CEA424-SV40 TAg transgenic mouse
The origin of the cells from which GEP-NETs arise is poorly understood. The transformation may take place either on precursor/stem cells or terminally differentiated neuroendocrine cells. There is a theory which describes the transformative events in the development of GEP-NETs. It postulates that the earlier the damage is, the poorer the prognosis is. In other word, damages occuring at a committed neuroendocrine precursor stage are more likely to induce well-differentiated NETs (NET-G1) while damages occuring early in stem cell progress may probably lead to the development of high grade or poorly differentiated neuroendocrine carcinomas (NECs)42.
Based on the mophology and the Ki-67 index, the gastric tumors developed in the CEA424-SV40 TAg transgenic mice were defined as neuroendocrine carcinomas. According to the theory described above, the tumor initiating cells may be the neuroendocrine precursor/stem cells. In addition, the tumor cells express multiple neuroendocrine markers and could produce several peptide, which further indicates that the transformation may have taken place in an early stage.
Gut neuroendocrine cells exist throughout the length of the gut and are the largest population of hormone-producing cells in the body3. At present, there have been at least 17 different neuroendocrine cells identified in the gastrointestinal tract including the pancreas which derive from local multipotent gastrointestinal stem cells, although in the past they are thought to migrate from the neural crest42, 156. Therefor, the newly described stem cell models in the gastrointestinal system may give some indications
where the tumor cells in the CEA424-SV40 TAg transgenic mouse model come from. Nick Barker et al described a population of Lgr5+ve cells, which are predominantly restricted to the base of adult pyloric glands, as multi-potent, self-renewing stem cells responsible for the long-term renewal of the gastric epithelium84. And it has been reported that Lgr5 positive cells can give rise to chromogranin A expressing cells123. To see if these Lgr5+ve cells might be the tumor initiating cells targeted by TAg, CEA424-SV40 TAgLgr5-EGFP-IRES-CreERT2 double transgenic mouse line was generated. However, there was no GFP/TAg double-positive cells observed in the gastric mucosa or in gastric tumors even in young mice. Lineage tracing experiment could not provide supportive information for the hypothesis that the Lgr5+ve cells may give rise to the tumor either (data from E. Vetter and W. Zimmermann, unpublished results). It seems unlikely that Lgr5+ve cells in the stomach could be the primary target of TAg.
Both the stomach and the small intestine are of common endodermal origin and have a constantly renewing epithelium. Moreover, the gastric units are somehow similar to the crypts in the small intestine with “stem cell zone” at the bottom. Since the stem cell theories for stomach and small intestine share some common ground and the stem cell markers of small intestine are better described, the stem cell models for the small intestine were used for reference. There are currently two epithelial stem cell pools described in the small intestine, the crypt base columnar (CBC) cells pool and the “+4” cell pool. As the typical “CBC” marker Lgr5 is not likely to be expressed in the tumor originating cells of the CEA424-SV40 TAg gastric tumor, the “+4” cell pool was taken into consideration. Bmi1 is the most prominent marker for the “+4” cells and is the most highly expressed epithelial stem cell marker tested in the cell lines derived from the CEA424-SV40 TAg gastric tumors. The tumor cell lines derived from the tumor showed a high expression of Bmi1 in RT-PCR and cDNA expression array. This could be further substantiated by western-blotting and immunohistochemistry. Consequently, Bmi1 message could also be found in the
for Bmi1 revealed TAg and Bmi1 double positive cells in the tumor tissue while a 100% positive rate was observed in the isolated cell lines. In addition, when SV40-TAg expression was knocked down by siRNA assay, the expression level of Bmi1 in cell line 424GC also went down,thus showing the interrelationship.
These observations indicate that these Bmi1 positive cells may be one target of the transformation induced by TAg. In fact, when we take a close look at Bmi1 function and the signaling pathway in which it is involved, more supportive information came up. Bmi1 is a component of polycomb group (PcG) multiprotein PRC1-like complex157. There have been studies which characterized the role of Bmi1 in multiple biological processes. For example, Bmi1 is involved in the self-renewal, maintenance and differentiation of normal stem cells in intestine95, lung158, liver159, prostate160, pancreas161, as well as in neural and hemopoietic system162, 163. It is found to be frequently up-regulated in various types of human cancers, including bladder cancer164, breast cancer165, gastric cancer166-169, liver cancer170, lymphoma171, prostate cancer160, 172 and skin cancer173, and is responsible for chemoresistance157, 174-178 . Except for contributing to epithelial-mesenchymal transition (EMT)179-181 and senescence regulation182, Bmi1 also takes part in the maintenance of “cancer stem cell” in some tumor types159, 183-186. Bmi1 performs these functions by regulating p16Ink4a and p19Arf genes. p16Ink4a regulates the formation of cyclin D/Cdk4/6 complex. This complex can phosphorylate RB1, thus promoting transcriptional activation of E2F1-regulated S-phase genes. On the other hand, p19Arf helps to maintain a certain level of p53 through inhibiting MDM2-mediated p53 degradation182. By controlling the activation of p16Ink4a and p19Arf, Bmi1 plays an essential role in cell cycle progression and DNA synthesis. It is worth noting that through these two pathways Bmi1 has an impact on RB1 and p53 indirectly, which are two major targets of TAg. This may indicate a closer internal relationship between Bmi1 and the CEA424-SV40 TAg gastric tumor. Our data clearly show that Bmi1 declines after inhibition of the TAg expression. So in the model presented here, TAg drives the expression of Bmi1 which may lead to a further enhancement of the TAg effects on RB1 and p53. On the
other hand, we observed that the TAg expression was not strongly affected by the knockdown of Bmi1. Therefore, the Bmi1 expression is more likely a consequence of TAg expression rather than the other way around.
In addition to epithelial stem cells, the possibility of mesenchymal stem cells as tumor originating cell in the CEA424-SV40 TAg transgenic mouse model was also presented in this thesis. The observation of the TAg positive cells in the muscularis mucosae of antrum and duodenum in young mice suggested a possible mesenchymal origin of the tumor. The expression of Etv1 in both the cell lines and the tumor tissue added more supportive information for this hypothesis. The more interesting observation was that, Etv1 expression level in the tumor tissue increased over the time course of the tumor development, while no significant difference of Bmi1 expression was observed between different age groups. Since the CEA-promoter, which controled the expression of TAg in the transgenic mice, contains Etv1 binding sites (A/CGGAAG), it is tempting to speculate that this may drive the transcription of the