The biological significance of a known normal and cancer stem cell marker CD133 remains elusive. We now demonstrate that the phosphorylation of tyrosine-828 residue in CD133 C-terminal cytoplasmic domain mediates direct interaction between CD133 and phosphoinositide 3-kinase (PI3K) 85 kDa regulatory subunit (p85), resulting in preferential activation of PI3K/protein kinase B (Akt) pathway in glioma stem cell (GSC) relative to matched nonstem cell. CD133 knockdown potently inhibits the activity of PI3K/Akt pathway with an accompanying reduction in the self-renewal and tumorigenicity of GSC. The inhibitory effects of CD133 knockdown could be completely rescued by expression of WT CD133, but not its p85-binding deficient Y828F mutant. Analysis of glioma samples reveals that CD133 Y828 phosphorylation level is correlated with histopathological grade and overlaps with Akt activation. Our results identify the CD133/PI3K/Akt signaling axis, exploring the fundamental role of CD133 in glioma stem cell behavior.

One of the serious sequelae of chronic hepatitis B virus (HBV) infection is hepatocellular carcinoma (HCC). Among all the proteins encoded by the HBV genome, hepatitis B virus X protein (HBx) is highly associated with the development of HCC. Although Notch1 signaling has been found to exert a tumor-suppressive function during HCC development, the mechanism of interaction between HBx expression and Notch1 signaling needs to be explored. In this study, we report that HBx expression in hepatic and hepatoma cells resulted in decreased endogenous protein levels of Notch1 intracellular domain (ICN1) and messenger RNA levels of its downstream target genes. These effects were due to a reduction of Notch1 cleavage by HBx through the suppression of presenilin1 (Psen1) transcription rather than inhibition of Notch1 transcription or its ligands' expression. Through transient HBx expression, decreased ICN1 resulted in enhanced cell proliferation, induced G1-S cell cycle progression, and blunted cellular senescence in vitro. Furthermore, the effect of blunted senescence-like growth arrest by stable HBx expression through suppression of ICN1 was shown in a nude mouse xenograft transplantation model. The correlation of inhibited Psen1-dependent Notch1 signaling and blunted senescence-like growth arrest was also observed in HBV-associated HCC patient tumor samples. Conclusion: Our results reveal a novel function of HBx in blunting senescence-like growth arrest by decreasing Notch1 signaling, which could be a putative molecular mechanism mediating HBV-associated hepatocarcinogenesis. (HEPATOLOGY 2010;52:142-154) H epatocellular carcinoma (HCC) is the fifth most common neoplasm and the third leading cause of cancer-related death in humans, with nearly 600,000 deaths annually worldwide. 1,2 Chronic hepatitis B virus (HBV) infection has been identified as a major risk factor for the development of HCC, especially in southeastern Asia and sub-Saharan Africa. [3][4][5] Several processes are involved in the development of HBV-associated hepatocellular carcinoma, including integration of HBV genes into host cell genome, sustained cycles of necrosis-inflammation-regeneration, activation of oncogenic pathways, and inactiAbbreviations: BrdU, 5-bromo-2 0 -deoxyuridine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; HCC, hepatocellular carcinoma; ICN1, Notch1 intracellular domain; mRNA, messenger RNA; NEXT1, extracellular truncated Notch1; Notch1-FL, full-length Notch1; qRT-PCR, quantitative real-time polymerase chain reaction; Psen1, presenilin1; Psen2, presenilin2; SA-b-gal, senescence-associated b-galactosidase; SD, standard deviation; TACE, tumor necrosis factor-a-converting enzyme.From the

CD133, a widely known cancer stem cell marker, has been proved to promote tumor metastasis. However, the mechanism by which CD133 regulates metastasis remains largely unknown. Here, we report that CD133 knockdown inhibits cancer cell migration, and CD133 overexpression promotes cell migration. CD133 expression is beneficial to activate the Src-focal adhesion kinase (FAK) signaling pathway. Further studies show that CD133 could interact with Src, and the region between amino acids 845 and 857 in the CD133 C-terminal domain is indispensable for its interaction with Src. The interaction activates Src to phosphorylate its substrate FAK and to promote cell migration. Likewise, a Src binding-deficient CD133 mutant loses the abilities to increase Src and FAK phosphorylation and to promote cell migration. Inhibition of Src activity by PP2, a known Src activity inhibitor, could block the activation of FAK phosphorylation and cell migration induced by CD133. In summary, our data suggest that activation of FAK by the interaction between CD133 and Src promotes cell migration, providing clues to understand the migratory mechanism of CD133 ؉ tumor cells.Tumor metastasis is a process by which tumor cells detach from the primary tumor into the stroma and invade blood vessels. This is followed by a secondary tumor growth at a distant site (1). This accounts for over 90% of lethality in cancer patients (2). Increasing evidence indicates that there is a subset of tumor cells contributing to metastasis that has properties of cancer stem cells (3). CD133, a pentaspan transmembrane glycoprotein, has rapidly gained clinical value with its wide use as a cancer stem cell marker (4 -13). A series of studies have shown that CD133 could directly regulate tumorigenesis, cell self-renewal, and angiogenesis (14 -16). Increasing evidence has also shown that CD133 is related to tumor metastasis in a variety of solid tumors (17)(18)(19)(20). For instance, activation of AKT/protein kinase B (PKB) signal pathway promotes invasion and migration of CD133 ϩ cancer stem cells in brain cancer (21). G protein-coupled receptor 87 was found to promote the growth and metastasis of CD133 ϩ cancer stem cells in hepatocellular carcinoma. CD133 expression is correlated with lymph node metastasis in pancreatic cancer (22,23). However, the mechanisms of CD133 regulating metastasis directly remain largely unknown.In head and neck cancer, CD133 could regulate Src kinase activity. The CD133/Src axis mediates tumor-initiating property and epithelial mesenchymal transition (24). Src is a member of the Src family of kinases, which is a non-receptor tyrosine kinase family (25,26). Src is highly expressed or highly activated in various human cancers (27). For instance, Src is highly active in glioblastoma multiforme specimens, and it represents an effective target for CD133 ϩ glioblastoma multiforme stem cell migration (28). Among the kinase substrates of Src, FAK 4 is an important mediator of tumor progressions and metastasis through the modulation of tumor cell migrat...

BCL2L12, a newly identified member of Bcl-2 family, contains a BH2 domain and a putative BH3 domain. It was found to be highly expressed in normal breast tissues, and was associated with favorable prognosis in breast cancer patients. Here, we reported that the mRNA levels of BCL2L12 and its transcript variant BCL2L12A could be upregulated upon cisplatin treatment in MDA-MB-231 breast cancer cells. Knockdown of BCL2L12 and BCL2L12A dramatically inhibited cisplatin-induced apoptosis. In contrast, ectopic expressions of each of the proteins promoted cisplatin-induced apoptosis. These results indicated that decreased expressions or loss of BCL2L12 and BCL2L12A may contribute to the cisplatin resistance in breast cancer patients. Furthermore, we found that cisplatin-induced downregulation of beta-catenin was partially suppressed in BCL2L12- and BCL2L12A-knocked down MDA-MB-231 cells, which indicated that knockdown of these two proteins may stabilize beta-catenin in cisplatin-induced apoptosis. In short, we proposed that BCL2L12 and BCL2L12A may play an important role in cisplatin-induced apoptosis in MDA-MB-231 breast cancer cells.

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