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CD133 is a universal marker of tissue stem/progenitor cells as well as cancer stem cells, but its physiological significance remains to be elucidated. Here we examined the relationship between expression of CD133 and features of gastric epithelial cells, and found that CD133-positive (CD133[+]) tumor cell lines formed well-differentiated tumors while CD133-negative (CD133[−]) lines formed poorly differentiated ones when subcutaneously injected into nude mice. We also found that CD133(+) and CD133(−) cell populations co-existed in some cell lines. FACS analysis showed that CD133(+) cells were mother cells because CD133(+) cells formed both CD133(+) and CD133(−) cells, but CD133(−) cells did not form CD133(+) cells. In these cell lines, CD133(+) cells formed well-differentiated tumors while CD133(−) cells formed poorly differentiated ones. In human gastric cancers, CD133 was exclusively expressed on the luminal surface membrane of gland-forming cells, and it was never found on poorly differentiated diffuse-type cells. Considering that poorly differentiated tumors often develop from well-differentiated tumors during tumor progression, these results suggest that loss of expression of CD133 might be related to gastric tumor progression. Microarray analysis showed that CD133(+) cells specifically expressed Sox17, a tumor suppressor in gastric carcinogenesis. Forced expression of SOX17 induced expression of CD133 in CD133(−) cells, and reduction of SOX17 caused by siRNA in CD133(+) cells induced a reduction in the level of CD133. These results indicate that Sox17 might be a key transcription factor controlling CD133 expression, and that it might also play a role in the control of gastric tumor progression. (Cancer Sci 2011; 102: 1313–1321)
Gastric adenocarcinomas are the second leading cause of cancer-related mortality in the world.(1) It is well established that environmental factors including Helicobacter pylori infection(2) and excessive intake of salt(3) are associated with tumorigenesis, but the molecular mechanism regulating gastric epithelial growth and differentiation has not been fully elucidated. Pathologically, gastric cancer can be divided into well-differentiated (intestinal) and poorly differentiated (diffuse) types according to the presence or absence of tubular structures.(4) The former is considered to occur through a stepwise change from chronic gastritis to atrophic gastritis, followed by spasmolytic polypeptide-expressing metaplasia and intestinal metaplasia, resulting in dysplasia and carcinomas,(5) but the latter through a less well-characterized sequence of events including a dysfunction of E-cadherin.(6)
Gastric cancers show great histological diversity, which tends to increase with deeper invasion and increased tumor diameter.(7) Thus, it is usual that they include a mixture of the well-differentiated and poorly differentiated types. In such cases, they are classified based on the predominant histological type according to the Japanese Classification of Gastric Carcinoma.(8) It has been a big problem how such mixed-type cancers are formed, and how it is related to the progression of gastric cancers. By detailed analysis of early and advanced gastric cancers, Ikeda et al.(9) suggested that the predominant histological type changed from well-differentiated to poorly differentiated types with the progression of tumors. On analysis of early gastric cancers, Saito et al.(10) indicated that small well-differentiated adenocarcinomas transformed into poorly differentiated tumors during their progression. Consistent with these, Takizawa and Koike(11) presented evidence showing that poorly differentiated tumor cells (signet ring cells) were formed from well-differentiated gastric carcinomas. Thus, it is probable that these two tumor types are related, and that some poorly differentiated tumor cells are derived from well-differentiated tumors through an unknown mechanism. It remains to be determined by what mechanism poorly differentiated tumor cells are induced from well-differentiated tumors. It has been difficult to analyze because no experimental systems have been reported in which the characteristics of gastric epithelial cells change from well-differentiated to poorly differentiated types in vitro.
We previously reported that Runx3, a runt domain transcription factor, is a major growth regulator of gastric epithelial cells, and that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.(12) We have so far established several Runx3−/−p53−/− gastric epithelial cell lines, all of which are tumorigenic, and some of which exhibit intestinal-type differentiation in vivo(13) and in vitro.(14) It remains to be solved how their differentiation is regulated, and whether changes in their differentiation are related to the progression of tumors.
CD133 is a transmembrane glycoprotein first identified on human hematopoietic stem cells by using a monoclonal antibody, AC133. Its murine counterpart, Prominin-1, has also been found on embryonic neuroepithelia and various other epithelia in embryos.(15) Recently, CD133/Prominin-1 (CD133) has been reported to be a marker of cancer stem cells in brain,(16,17) lung,(18) pancreas,(19) prostate,(20) colon(21,22) and liver(23) tumors. However, controversial results have been reported regarding the significance of CD133 in gastric tumorigenesis since Smith et al.(24) reported that 47–55% of gastric tumor samples were positive for CD133, while Boeg and Prinz(25) found that expression of CD133 decreased with the progression of carcinogenesis in gastric cancers.
Here, we examined the relationship between expression of CD133 and the differentiation potency of Runx3−/−p53−/− gastric epithelial cells, and obtained evidence that loss of CD133 expression might be related to the phenotypic change of tumors from well-differentiated to poorly differentiated ones. Our analysis also indicated that Sox17, a key regulator of the development of a definitive endoderm(26,27) might be involved in the control of CD133 expression in gastric epithelial cells.
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In the present study, we examined the roles of CD133 in the growth and differentiation of gastric epithelial cells. We could not obtain any evidence indicating that CD133 expression is related to their growth, but we obtained evidence suggesting that it is involved in gastric epithelial morphogenesis. As shown in Table 1, CD133 expression was significantly correlated to the formation of glandular structures. Moreover, we found that in two cell lines in which CD133(+) and CD133(−) cells co-existed, CD133(+) cells formed glandular structures while CD133(−) cells did not, both in vivo and in vitro. These results strongly suggest that CD133 expression and gastric epithelial morphogenesis are controlled through a common mechanism. It has been reported that most human gastric cancer cell lines do not express CD133,(32) and that the morphological characteristics of human gastric carcinomas gradually decrease during passages in vitro.(31) Our results are consistent with these, and suggest that the mechanism underlying epithelial morphogenesis might be elucidated by determining how the expression of CD133 is regulated. Boeg and Prinz(25) reported that CD133 expression was reduced in advanced stages of gastric cancer. This might be explained by our finding that CD133 is expressed on well-differentiated-type gastric tumor cells, but not on poorly differentiated-type tumor cells, which are usually found in advanced stages. We found that CD133 was exclusively expressed by glandular cells but not by diffuse-type cells in human gastric cancers (Fig. 4). Similar results have been reported in colorectal tumors,(29,34) ductal adenocarcinomas of the pancreas(35) and esophageal adenocarcinomas,(36) although Zhao et al.(37) reported that CD133 was also expressed by cells at invasive depth.
In the present investigation, we found that CD133(+) cells are the mother cells for both CD133(+) and CD133(−) cells, and that CD133(−) cells did not form glandular structures in the absence of CD133(+) cells for both GIF-5 and GIF-11 cells. When GIF-5 or GIF-11 cells were subcutaneously injected into nude mice they formed glandular structures in spite of that CD133(−) cells comprised the major and CD133(+) cells the minor population, indicating that CD133(+) cells induced glandular structure formation by CD133(−) cells. It is possible that CD133(+) cells secrete humoral factors and/or extracellular matrix, which induce glandular structure formation by CD133(−) cells. Elucidation of the mechanism is an important task for the future.
In cultures of GIF-5 and GIF-11 cells, CD133(+) cells formed both CD133(+) and CD133(−) cells, indicating that CD133 expression is easily lost during in vitro growth of these cells. If loss of CD133 expression is related to the loss of the morphogenetic ability of the cells, loss of CD133 expression might be observed during the progression of gastric cancers when poorly differentiated-type tumors are formed from well-differentiated types. The mechanism of progression has not been fully elucidated, although the methylation of tumor suppressor genes,(38) secretion of growth factors,(39) epithelial–mesenchymal transition,(40) and fibroblast-derived proteases(41) have been demonstrated to be involved in it. Thus, GIF-5 and GIF-11 cells might be good models for studying the mechanism of gastric tumor progression.
CD133 is widely used for the identification of cancer stem cells in various tumors, but its physiological significance is not fully understood, partly because CD133-knockout mice can survive without major defects, although progressive degeneration of mature photoreceptors has been found in these mice.(42) It is probable that expression of CD133 is not directly related to the function of stem cells, but that its expression is controlled through a mechanism that is related to the function of stem cells. Thus, it would be important to elucidate the mechanism regulating the expression of CD133, because it might function in the regulation of the identity of stem cells. Our system involving GIF-5 cells would be useful for future study to identify factors regulating CD133 expression.
To identify the factors, we compared the gene expression profiles of CD133(+) and CD133(−) cells and found that Sox17 was a candidate factor controlling expression of CD133. Forced expression of SOX17 in CD133(−) cells and reduction of SOX17 in CD133(+) cells showed that SOX17 regulated CD133 expression in both mouse and human gastric epithelial cells. Thus, we concluded that Sox17 is a key transcription factor controlling CD133 expression. It is probable that loss of Sox17 induces loss of CD133, which directly/indirectly results in the development of gastric tumors into more aggressive poorly differentiated types. In the present study, we immunohistochemically examined the relationship between expression of CD133 and that of SOX17 in human gastric tumors, but a strong correlation could not be found between them. It is possible that SOX17 is diffused during immunohistochemical procedures because it was found not only in the nuclei but also in the cytoplasm in the present study (Fig. 6). Better results might be obtained by improving the procedures. It is also possible that Sox17 functions in collaboration with other factors to regulate CD133 expression. It is necessary to identify these factors and to elucidate the mechanism to fully understand how gastric epithelial morphogenesis is controlled.
The function of SOX17 in gastric carcinogenesis is not fully understood. Katoh(43) reported that SOX17 was more preferentially expressed in the esophagus, stomach and small intestine than in the colon and rectum in normal gastrointestinal tract, and that it was almost undetectable in human cancer cell lines and also in 66 cases of human primary tumors derived from various tissues. Consistent with this, a tumor-suppressive role of SOX17 has been reported in colon carcinoma cells(44) and hepatocellular carcinomas.(45) Zhang et al.(46) reported that SOX17 silencing due to promoter hypermethylation was an early event during tumorigenesis in colorectal cancer. We also found that SOX17 is not expressed in most human gastric tumor cell lines including MKN45 and MKN74, and that this might be caused by promoter hypermethylation because its expression was significantly increased by treatment with 5-aza 2′-deoxycytidine in these cells (Shimada S., Fukamachi H. and Yuasa Y., unpublished data).
Recently, Du et al.(33) showed that Sox17 played a tumor suppressive role through suppression of Wnt signaling in mouse gastric carcinogenesis. They also suggested that Sox17 protected early tumors from malignant progression at an early stage of tumorigenesis, and that downregulation of Sox17 contributed to malignant progression through promotion of Wnt activity. These results are consistent with ours and suggest that loss of Sox17 induces gastric tumor progression through, at least in part, the transformation of well-differentiated-type tumors into poorly differentiated types.
In the present study, we could not obtain direct evidence that Sox17 regulates gastric epithelial morphogenesis, because we could not induce glandular structure formation by MKN45 or MKN74 cells, even when expression of SOX17 and CD133 was significantly elevated (data not shown). It is probable that not only SOX17 expression but also the expression of other genes might be necessary for the cells to form glandular structures. Gland-forming GIF-5 cells specifically expressed various genes that are related to cell attachment and extracellular matrix deposition (Table 2). Combinational expression of these genes might be necessary to induce glandular structure formation. Further studies are needed to elucidate the mechanism by which gastric epithelial morphogenesis is regulated.