M1978 CD133 Positive Cancer Stem-Like Cells Are Induced Through Notch Signaling Pathway in Poorly Differentiated Human Gastric Cancer

Abstract

Background & Aim: Cancer stem cells are thought to be stem-like phenotype of tumor cells that initiate and propagate tumor. CD133, a cell-surface glycoprotein, is considered as a putative cancer stem cell marker. In gastric cancer, diffuse-type (poorly differentiated) cancer cells are thought to arise from hyperplastic stem cells, which is different from intestinal type gastric cancer cells, but little is known about this molecular tumorigenesis mechanism in terms of cancer stem cells. In this study, we investigated whether there are CD133 positive tumor cells bearing characteristics of cancer stem cells in human gastric cancer, and the role of Notch signaling, which controls cell fate decisions and tumorigenesis with the regulation of self-renewal of stem cells.Methods: Eight gastric cancer cell lines were analyzed for the expression of CD133 and cleaved (activated) Notch1 by RT-PCR and by Western blotting. In addition, CD133 expression in 44 human gastric cancer tissues (17 intestinaltype and 27 diffuse-type) was assessed by immunohistochemistry. Next, after establishing Notch1 RNAi inducible stable clones in a gastric cancer cell line, we performed Notch1 knockdown experiments to investigate whether Notch1 signaling regulated the expression of CD133 by flow cytometry. Also, the proliferation of these cells was assessed by 3-(4,5dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the tumorigenesis of them was determined by soft agar assay. Results: Among eight cell lines we screened, we discovered that both CD133 and cleaved Notch1 were highly expressed in 4 poorly differentiated cell lines, especially in KATO III and NUGC-4 cells, but not in other 4 welldifferentiated cell lines. Similarly, immunohistochemistry of human gastric cancer tissues revealed that CD133 was expressed in 11(41%) of 27 diffuse-type (poorly differentiated) tissues, but not as high in intestinal-type tissues. Using the Notch1 knockdown inducible KATO III cells, a gastric cancer cell line highly expressing CD133, we found out that Notch1 blockade reduced CD133 positive cell fraction to almost half by flow cytometry. This reduction in Notch1 and CD133 turned out to suppress the proliferation (43.4 ± 3.3%) and tumor-forming capacity due to the depletion of the stem-like cells. Conclusions: These results suggest that in gastric cancer, CD133 positive cancer stem-like cells exist only in poorly differentiated cancer cells, and Notch1 signaling pathway contributes to the induction of these cells and subsequent tumor propagation. This may give us a clue to understand cancer stem cells in gastric cancers, which would lead us to a new therapy for them.