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

Androgen receptor (AR) is essential for the maintenance of the male reproductive systems and is critical for the carcinogenesis of human prostate cancers (PCas). D-type cyclins are closely related to the repression of AR function. It has been well documented that cyclin D1 inhibits AR function through multiple mechanisms, but the mechanism of how cyclin D3 exerts its repressive role in the AR signaling pathway remains to be identified. In the present investigation, we demonstrate that cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11 p58 ) repressed AR transcriptional activity as measured by reporter assays of transformed cells and prostate-specific antigen expression in PCa cells. AR, cyclin D3, and CDK11 p58 formed a ternary complex in cells and were colocalized in the luminal epithelial layer of the prostate. AR activity is controlled by phosphorylation at specific sites. We found that AR was phosphorylated at Ser-308 by cyclin D3/CDK11 p58 in vitro and in vivo, leading to the repressed activity of AR transcriptional activation unit 1 (TAU1). Furthermore, androgen-dependent proliferation of PCa cells was inhibited by cyclin D3/CDK11 p58 through AR repression. These data suggest that cyclin D3/CDK11 p58 signaling is involved in the negative regulation of AR function.Androgen receptor (AR), a member of the nuclear receptor family, directly regulates patterns of gene expression in response to the steroids testosterone and dihydrotestosterone (DHT) and is subsequently involved in the regulation of the development and differentiation of the male reproductive system (10). Similar to other steroid receptors, AR contains a transactivation domain (TAD), also named AF-1, in its N terminus, a ligand binding domain (LBD) in its C terminus, a DNA-binding domain (DBD), and a hinge region between the TAD and LBD. The transcriptional activation unit 1 (TAU1) and TAU5 motifs in the AR N-terminal domain (NTD) (residues 101 to 370 and 360 to 485, respectively) as well as the AF-2 motif in the AR LBD have been implicated in directly contacting p160 proteins and mediating transcription (1,23,31,48).AR is a phosphoprotein whose function is regulated by the modulation of its phosphorylation status at different sites (4). The consensus phosphorylation sites found in AR indicate that AR could be a substrate for DNA-dependent kinase, protein kinase A, protein kinase C, mitogen-activated protein kinase, and casein kinase 2 (4). Ser-16, Ser-81, Ser-94, Ser-256, Ser-308, Ser-424, and Ser-650 have been identified as being phosphorylation sites of AR by mutagenesis, peptide mapping, and mass spectrometry (6, 60). Recently, several Ser/Thr protein kinases have been found to phosphorylate AR at the abovementioned sites in vitro and in vivo. For example, AR Ser-515 is phosphorylated by mitogen-activated protein kinase, Ser-213 and Ser-791 are phosphorylated by Akt, and Ser-650 is phosphorylated by p38␣ and JNK1 (17,30,57).More and more studies suggest that cyclins and cyclin-dependent kinases (CDKs) are also involved in th...

Background Whether tumour-infiltrating lymphocytes (TILs) play different roles in different molecular subtypes of breast cancer remains unknown. Additionally, their prognostic and predictive value in different molecular subtypes of breast cancer is still controversial. The aim of our meta-analysis was to assess the prognostic and predictive value of TILs in different molecular subtypes of breast cancer by summarizing all relevant studies performing multivariate analysis. Methods PubMed, Embase, EBSCO, ScienceDirect, the Cochrane Database and Web of Science were comprehensively searched (until March 2020). Hazard ratios (HRs), odds ratios (ORs) and their 95% confidence intervals (CIs) were used as effect measures to perform our meta-analysis. A random effect model was used. Stata software, version 15 (2017) (StataCorp, College Station, TX, USA) was used to perform the statistical analysis. Results Thirty-three studies including 18,170 eligible breast cancer patients were analysed. The meta-analysis showed that high TIL expression was significantly associated with increased pathological complete response (pCR) rates after neoadjuvant chemotherapy in patients with the HER2-enriched molecular subtype (OR = 1.137, 95% CI [1.061 ~ 1.218], p < 0.001) and triple-negative breast cancer (TNBC) subtype (OR = 1.120, 95% CI [1.061 ~ 1.182], p < 0.001). However, high TIL expression was not significantly associated with high pCR rates after neoadjuvant chemotherapy in patients with the luminal molecular subtype of breast cancer (OR = 1.154, 95% CI [0.789 ~ 1.690], p = 0.460). We carried out a meta-analysis on the HRs of overall survival (OS) and disease-free survival (DFS) to assess the prognostic value of TILs in breast cancer with different molecular subtypes more deeply. Our meta-analysis confirmed that high TILs were associated with significantly improved DFS in patients with the HER2-enriched molecular subtype [HR = 0.940, 95% CI (0.903 ~ 0.979), p = 0.003] and TNBC molecular subtype [HR = 0.907, 95% CI (0.862 ~ 0.954), p < 0.001]. However, high TILs were not associated with significantly better DFS in patients with the luminal molecular subtype of breast cancer [HR = 0.998, 95% CI (0.977 ~ 1.019), p = 0.840]. Furthermore, the results confirmed that high TILs were significantly related to better OS in patients with the HER2-enriched molecular subtype [HR = 0.910, 95% CI (0.866 ~ 0.957), p < 0.001] and TNBC molecular subtype [HR = 0.869, 95% CI (0.836 ~ 0.904), p < 0.001]. Conversely, the summarized results indicated that high TILs were significantly associated with poor OS in patients with the luminal molecular subtype of breast cancer [HR = 1.077, 95% CI (1.016 ~ 1.141), p = 0.012]. Conclusions Our meta-analysis confirms that high TILs are associated with favourable survival and predicts pCR in breast cancer patients with the TNBC and HER2-enriched molecular subtypes.

The elevated levels of ␤1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5-region flanking the transcription start point of the GalT I gene (؊1653 to ؉52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides ؊205 and ؊200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the ␤1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.The enzyme ␤1,4-galactosyltransferase I (GalT I 1 ; EC 2.4.1.38) is a constitutively expressed type II membrane-bound glycoprotein in vertebrates (1). It is unusual that it resides in two distinct subcellular compartments, the trans-Golgi network and the cell surface (2, 3). In the trans-Golgi complex, GalT I is one of the key enzymes involved in the sugar chain synthesis that catalyzes the transfer of galactose from UDP-Gal to terminal N-acetylglucosamine, forming the Gal␤134GlcNAc structure (4). Cell surface GalT I acts as a recognition molecule and participates in a number of cellular interactions, including neurite extension, cell growth, spermegg interaction, cell spreading, and migration (5-9).Neoplasms undergo various changes in the carbohydrate moieties of their glycoconjugates, which indicate that the glycosyltransferases themselves may change in malignancies. Consistent with this hypothesis, the importance of specific sialyltransferases, fucosyltransferases, N-acetylglucosaminyltransferase in tumorigenesis, and metastasis has been demonstrated (10 -12).Although the precise role of oligosaccharides in metastasis is presently unknown, accumulated evidence has shown that a number of highly metastatic murine and human cell lines are ...

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...

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